Conjugates of biologically active molecules to functionalized polymers

ABSTRACT

This document relates to conjugates of a biologically active molecule or a derivative thereof and functionalized (e.g., mono- or bi-functional) polymers (e.g., polyethylene glycol and related polymers) as well as methods and materials for making and using such conjugates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application under 35 U.S.C. § 371of International Application No. PCT/IB2013/001885, having aninternational filing date of Jun. 12, 2013, which claims priority toU.S. Provisional Ser. No. 61/658,827, filed Jun. 12, 2012; 61/785,996,filed Mar. 14, 2013; 61/658,839, filed Jun. 12, 2012; 61/786,121, filedMar. 14, 2013; 61/658,835, filed Jun. 12, 2012; 61/786,162, filed Mar.14, 2013; 61/658,836, filed Jun. 12, 2012; 61/786,237, filed Mar. 14,2013; 61/658,850, filed Jun. 12, 2012; 61/786,221, filed Mar. 14, 2013;61/658,853, filed Jun. 12, 2012; 61/786,265, filed Mar. 14, 2013;61/658,856, filed Jun. 12, 2012; and 61/786,287, filed Mar. 14, 2013;all of which are incorporated by reference in their entireties.

TECHNICAL FIELD

This document relates to conjugates of a biologically active molecule toa functionalized (e.g., mono- or bi-functional) polymers (e.g.,polyethylene glycol and related polymers) as well as methods andmaterials for making and using such conjugates.

BACKGROUND

Pharmacokinetic and immune stimulating properties of proteins andsynthetic drugs may be controlled by their conjugation to certainpolymers. For example, polyethylene glycol (PEG) can be conjugated toproteins to achieve this effect (Fee and Van Alstine, ChemicalEngineering Science, 61:924-934 (2006)). Such conjugation can take placeif the relatively non-reactive hydroxyl groups present in PEG moleculesare substituted by other, more reactive moieties (Jagur-Grudzinski,Reactive & Functional Polymers, 39:99-138 (1999)). A standard, linearPEG molecule is chemically a diol, which could suggest that the processof PEG derivatization and purification of products should be trivial.However, the polymeric nature of this diol, together with itsamphiphilic properties can make these manipulations difficult. In somecases, the typical laboratory process for separation of difficultreaction mixtures, silica gel-based flash column chromatography, canfail for PEG with molecular weight higher than 1000. Neithercrystallization nor precipitation appear adequate to achieve separationof PEG-containing materials, even if these methods can be used forefficient removal of other, contaminating substances with low molecularweight. Most reaction mixtures containing modified PEG molecules lack areliable analytical method to control or to prove their composition.Polymers with functions that influence only minimally the hydrophobicproperties of the polymer can be difficult to analyze by chromatography.The same applies for polymers with functions carrying only a minimalcharge. This also applies for preparative chromatographic separation ofcharged polymers as described elsewhere for the separation of mono- anddi-carboxyl modified PEG molecules (Drioli et al., Reactive & FunctionalPolymers, 48:119-128 (2001)).

Confirmation of results of the synthesis based on NMR can be useless, aslong as one is not sure about the purity of the product, and this istypically only obtained by chromatographic methods. This unusualconclusion comes from observations that an equimolar mixture ofnon-derivatized polymer and bis-derivatized polymer will produce an NMRpattern identical to the pure mono-derivatized polymer. Massspectrometry can be complicated since most PEG exists not in the form ofa single component, but is rather a Gaussian population of differentpolymer lengths, centered on its average molecular weight. Thus, even ifall distinct components of the same type should have their massincreased by the same factor, the presence of unreacted and bis-modifiedmaterial can obscure the picture of the analysis. The literaturediscusses this problem only sporadically, and often nothing is mentionedabout analysis of the product or its purification. Many authors make theimpression that the process that they describe is ongoing withquantitative yield, and thus the quality of the product does not need tobe analyzed or questioned. This non-scientific approach can befrequently encountered in the chemistry of PEG. There are many examplesin the literature presenting synthetic procedures with four to fiveconsecutive steps without a single analysis of the product at any ofthese steps, without any attempts to purifying the product, and assuming100 percent purity at the end of the process. It is, therefore, notstrange that researchers after closer testing question these productsand their purity (Ananda et al., Anal. Biochem., 374, 231-242 (2008)). Acommonly accepted escape from the problem of selective modification isto work with a polymer that has one end blocked from the beginning by astable chemical group, most often a methoxy group (mPEG). In theory,this blockage converts a PEG molecule to a monofunctional compound, andas such, it could be fully converted to the second derivatized form byincreasing the amount of derivatizing reagent and/or time for reaction.Unfortunately, many of reactions commonly applied for derivatization ofPEG are sluggish and only seldom go to completion. On the other hand,mPEG preparations contain significant percentages of PEG diol component.Moreover, the amount of this contamination increases with the length ofmPEG, and this contamination can be hard to avoid. Consequently,derivatization will also result in formation of symmetrical,bis-derivatizated PEG, and its presence in the conjugating mixtureresults in formation of cross-linked products with unknown pharmacologicproperties or a possible loss of protein activity. Therefore, pure,monofunctional polymers are usually preferred for protein modification,but one should be aware that purification of mPEG from its diol PEGcontamination is practically impossible.

Nearly all of existing reactions, used today for derivatization of PEG,belong either to the alkylation-based or the acylation-based category.In the first case, the alkoxy anion, generated from PEG, is reactingwith incoming electrophilic modifying reagent. Eventually, the activatedPEG, subjected with a good leaving group, is itself an object of anucleophilic attack. To this category belong processes resulting inthiolation, amination, azidation, and introduction of a carboxyl or analdehyde group. Modified PEG's of this category will have theirfunctional group connected directly to the PEG terminal carbon atom orthese groups will be linked via an ether bond, a thioether bond, or asecondary amino group.

The second category, acylation, is based on a nucleophilic reaction ofPEG's hydroxyl, (or another group present in a modified PEG—often anamino group), on an incoming acylating reagent. In many cases, thisfirst acylation is followed by a second acylation that actuallyintroduces the modification of interest to the PEG molecule. Functionalgroups incorporated by this method can be linked to the rest of PEG byan amido, a carbamido, urethane, thiourethane, or a simple ester group.These linking groups and the chemistry behind them belong to the verytraditional methods of combining two chemical identities.

Polyethylene glycols (PEG) coupled to phosphoramidites are used fordirect coupling of PEG molecules to synthetic nucleic acids. One exampleis4,4′-dimethoxytrityl-polyethyleneglycol-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.In these compounds, the phosphoramidite group is the part of thereactive functionality for linking the compound to a synthetic nucleicacid. It is designed to work in a completely water-free environment: Inthe presence of water, the phosphoramidite group can decomposeinstantaneously, making such PEG phosphoramidites inappropriate forconjugation to biological material in water-containing or aqueoussolution. In particular, these PEG phosphoramidites can be inappropriatefor conjugation to biological substances which are not soluble, stableor sufficiently reactive in non-aqueous media. Furthermore, alreadymildly acidic biological substances can decompose these PEGphosphoramidites. Finally, these PEG phosphoramidites contain a labileprotecting group adjacent to the phosphorous atom which is speciallydesigned to convert the intermediate phosphotriester to aphosphodiester. Phosphodiesters can be readily degraded enzymatically invivo.

SUMMARY

This document provides conjugates of a biologically active molecule(e.g., TNF inhibitors, insulin, omalizumab, clotting factors,polypeptides that boost red or whit blood cell production, an antibody),or derivatives thereof to functionalized (e.g., mono- or bi-functional)polymers (e.g., polyethylene glycol and related polymers) as well asmethods and materials for making and using such conjugates. Theconjugates described herein can have an altered pharmacokinetic andpharmacodynamic profile, including, for example, one or more of reduceddosage frequency, extended circulation time, and reduced antigenicproperties. The functionalized polymers include one or more linkinggroups selected from a phosphotriester, a phosphoramidate, athiophosphotriester, and a thiophosphoramidate. For example, abiologically active molecule or a derivative thereof can be conjugatedto a PEG polymer having at least one functional group at one terminuscovalently bound to the rest of the polymer via a phosphotriester orphosphoramidate linking group. A biologically active molecule or aderivative thereof can be conjugated to a functionalized polymerprovided herein that includes different linking groups at each of itstermini. Also provided herein is a biologically active molecule or aderivative thereof conjugated to a functionalized polymer in which atleast one terminus is modified with a blocking group (e.g., methoxygroup) and at least another terminus is functionalized with a linkinggroup as described herein.

As described herein, preparations of a functionalized polymer having oneor more functional groups linked as described herein can be obtained ina manner where greater than 50 percent by weight (e.g., greater than 75percent, greater than 80 percent, greater than 90 percent, greater than95 percent, greater than 98 percent, and greater than 99 percent byweight) of the preparation is the desired functionalized polymer freefrom contaminants and can be used to conjugate to a biologically activemolecule or a derivative thereof. As a person of ordinary skill in theart would understand, a functionalized polymer preparation includes aGaussian population of different polymer lengths centered on an averagemolecular weight, and such a functionalized population would not beconsidered contaminating. For example, a mono-functionalized PEGpopulation can be separated nearly quantitatively from contaminants ofPEG functionalization (e.g., unreacted PEG and poly-functionalized PEGpopulations). The separation of a functionalized polymer provided hereincan be facilitated through the use of a removable hydrophobic separationhandle (e.g., a substituted or unsubstituted trityl group) which uponremoval allows for preparative isolation of product (e.g., pureproduct), free or substantially free from unreacted polymer andpoly-functionalized polymer.

Having the ability to isolate functionalized polymers in high purity canallow chemists to more easily control subsequent reactions or the purityof the downstream products, e.g., conjugates to a biologically activemolecule or a derivative thereof. In some cases, having the ability tointroduce all functional groups through a unified process using similar,mild reagents and reaction conditions can allow for the production of afunctionalized polymer through a fast and nearly quantitative reaction.

The ability to isolate functionalized polymers quantitatively can allowchemists to more easily control subsequent reactions or the purity ofconjugates of a biologically active molecule such as a TNF inhibitor,insulin, omalizumab, a clotting factor, a polypeptide that boosts red orwhite cell production, or an antibody, coupled to a functionalizedpolymer provided herein.

A linking group, in addition to linking the functional group orseparation handle, can act as a linker between the polymer and abiologically active molecule or derivative thereof. For instance, afunctionalized polymer of high purity can be coupled to a biologicallyactive molecule or derivative thereof. In such conjugates, the linkercan form covalent bonds to the polymer and a biologically activemolecule or derivative thereof. The coupling can take place in anaqueous reaction medium.

Furthermore, the linking groups provided herein are generally resistantto chemical and enzymatic degradation, providing for stable storage andincreased safety and efficacy in vivo.

In one aspect, this document features a method of making a biologicallyactive molecule conjugate. The method includes reacting a biologicallyactive molecule or a derivative thereof with a preparation comprising awater-soluble, non-peptidic, and non-nucleotidic polymer backbone havingat least one terminus covalently bonded to a structure of formula (1)under conditions suitable for group M to react with a biologicallyactive molecule or the derivative thereof:

or a salt thereof, wherein A is the point of covalent bonding to theterminus of the polymer backbone; E is O or S; K is selected from thegroup consisting of: alkylene, alkyleneoxyalkylene, and oligomericalkyleneoxyalkylene; G is selected from the group consisting of:hydrogen, alkoxy, and a hydrophobic separation handle; Z¹ and Z² areindependently selected from O and NH, wherein only one of Z¹ and Z² canbe NH; L is selected from the group consisting of: a divalent radical ofa nucleoside, alkylene, alkyleneoxyalkylene, oligomericalkyleneoxyalkylene, and unsubstituted and substituted arylene; M is aprotected group that when deprotected is reactive with a biologicallyactive molecule or derivative thereof or is a group reactive with abiologically active molecule or derivative thereof; R is absent orselected from the group consisting of: hydrogen, a protecting group, ahydrophobic separation handle, or an activating group; R¹ is absent or ahydrophobic separation handle; wherein when M is a protected group thatwhen deprotected is reactive with a biologically active molecule orderivative thereof, then R is a protecting group or a hydrophobicseparation handle; wherein when M is a group reactive with abiologically active molecule or derivative thereof, R is absent,hydrogen, or an activating group; and wherein only one of R, R¹, and Gcan be a hydrophobic separation handle.In some embodiments, the polymer backbone has from 2 to 100 termini. Insome embodiments, only one termini of the polymer backbone is covalentlybonded to the structure of formula (1). In some embodiments, only onetermini of the polymer backbone is covalently bonded. In someembodiments, the polymer backbone has two termini. In some embodiments,only one termini of the polymer backbone is covalently bonded to thestructure of formula (1). In some embodiments, both termini of thepolymer backbone are covalently bonded to the structure of formula (1).

A polymer backbone can be selected from the group consisting ofpoly(alkylene glycol), poly(oxyethylated polyol), poly(olefinicalcohol), poly(α-hydroxy acid), poly(vinyl alcohol), polyoxazoline, andcopolymers. For example, a polymer backbone can be poly(ethyleneglycol). In some cases, a poly(ethylene glycol) has an average molecularweight from about 500 Da to about 100,000 Da.

In some embodiments, one of Z¹ and Z² is NH and the other is O. In someembodiments, Z¹ is O and Z² is NH. In some embodiments, Z¹ is NH and Z²is O. In some embodiments, both Z¹ and Z² are O.

The group reactive with a biologically active molecule or the derivativecan be selected from the group consisting of: hydroxyl, amine, thiol,carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide,acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide,dithiopyridine, and iodoacetamide.

In some embodiments, K is selected from the group consisting of:methylene, ethylene, propylene, isopropylene, butylene, isobutylene,sec-butylene, tert-butylene, and hexylene, or a residue from diethyleneglycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol.

In some embodiments, G is a substituted or unsubstituted trityloxy.

In some embodiments, L is a substituted or unsubstituted C₁-C₁₂alkylene.

In some embodiments, R is selected from the group consisting of: trityl,monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.

In some embodiments, a group reactive with a biologically activemolecule or the derivative is carboxyl and R is absent or selected fromthe group consisting of N-hydroxysuccinimidyl, p-nitrophenyl, orpentachlorophenyl.

In some embodiments, the preparation includes at least 50% by weight(e.g., at least 60%, 70%, 80%, 90%, 95%, or 98% by weight) of thewater-soluble, non-peptidic, and non-nucleotidic polymer backbone havingat least one terminus covalently bonded to a structure of formula (1).

In some embodiments, the method further can include (i) removing thehydrophobic separation handle(s) from the structure of formula (1) and(ii) optionally reacting the compound obtained in step (i) with anactivating agent before reacting with a biologically active molecule orthe derivative.

In some embodiments, the method further can include removing thehydrophobic separation handle(s) from the structure of formula (1) afterreacting with a biologically active molecule or the derivative.

This document also features a conjugate, or a pharmaceuticallyacceptable salt thereof, that includes a water-soluble, non-peptidic,and non-nucleotidic polymer backbone as in a structure of formula (9):

or a salt thereof,

wherein A is the point of covalent bonding to the terminus of thepolymer backbone; E is O or S; K is selected from the group consistingof: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; Gis selected from the group consisting of: hydrogen, alkoxy, and ahydrophobic separation handle; Z¹ and Z² are independently selected fromO and NH, wherein only one of Z¹ and Z² can be NH; L is selected fromthe group consisting of: a divalent radical of a nucleoside, alkylene,alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstitutedand substituted arylene; R¹ is absent or a hydrophobic separationhandle, wherein only one of R¹ and G can be a hydrophobic separationhandle; L² is a covalent linking moiety between L on the polymerbackbone and B; and B is a biologically active molecule or a derivativethereof.

This document also features a method of making a biologically activemolecule conjugate. The method includes reacting a biologically activemolecule or a derivative thereof with a preparation that includes acompound of formula (2) under conditions suitable for group M to reactwith a biologically active molecule or the derivative thereof:

or a salt form thereof, wherein polymer is a linear, water-soluble,non-peptidic, and non-nucleotidic polymer backbone, wherein each linkinggroup is bonded at a different terminus of the polymer; E and E¹ areindependently O or S; K and K₁ are independently selected from the groupconsisting of: alkylene, alkyleneoxyalkylene, and oligomericalkyleneoxyalkylene; G and G₁ are independently absent or are selectedfrom the group consisting of: alkoxy and a hydrophobic separationhandle; each pair of Z¹ and Z² and Z³ and Z⁴ are independently selectedfrom O and NH, wherein only one of each pair of Z¹ and Z² and Z³ and Z⁴can be NH; L and L¹ are independently selected from the group consistingof: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene,oligomeric alkyleneoxyalkylene, and unsubstituted and substitutedarylene; M and M¹ are independently selected from a protected group thatwhen deprotected is reactive with a biologically active molecule or thederivative or a group reactive with a biologically active molecule orthe derivative, wherein M and M¹ are different; and R and R¹ areindependently absent, hydrogen, a protecting group, or an activatinggroup; wherein when M is a protected group that when deprotected isreactive with a biologically active molecule or the derivative, then Ris a protecting group or a hydrophobic separation handle; wherein when Mis a group reactive with a biologically active molecule or thederivative, R is absent, hydrogen, or an activating group; wherein whenM¹ is a protected group that when deprotected is reactive with abiologically active molecule or the derivative, then R¹ is a protectinggroup or a hydrophobic separation handle; and wherein when M¹ is a groupreactive with a biologically active molecule or the derivative, R¹ isabsent, hydrogen, or an activating group.

In some embodiments, one of Z¹ and Z² is NH and the other is O. In someembodiments, Z¹ is O and Z² is NH. In some embodiments, Z¹ is NH and Z²is O. In some embodiments, both Z¹ and Z² are O. In some embodiments,one of Z³ and Z⁴ is NH and the other is O. In some embodiments, Z³ is Oand Z⁴ is NH. In some embodiments, Z³ is NH and Z⁴ is O. In someembodiments, both Z³ and Z⁴ are O.

In some embodiments, the group reactive with a biologically activemolecule or the derivative thereof is selected from the group consistingof: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl,alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide,aminoxy, maleimide, dithiopyridine, and iodoacetamide.

In some embodiments, K and K¹ are independently selected from the groupconsisting of: methylene, ethylene, propylene, isopropylene, butylene,isobutylene, sec-butylene, tert-butylene, and hexylene, or a residuefrom diethylene glycol, triethylene glycol, tetraethylene glycol orhexaethylene glycol.

In some embodiments, G and G¹ is independently selected from asubstituted or unsubstituted trityloxy.

In some embodiments, L and L¹ is independently a substituted orunsubstituted C₁-C₁₂ alkylene.

In some embodiments, R or R¹ is independently selected from the groupconsisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, cyclicacetal, and combinations of thereof.

In some embodiments, the polymer is poly(ethylene glycol). For example,a poly(ethylene glycol) having an average molecular weight from about500 Da to about 100,000 Da.

In some embodiments, the preparation includes at least 50% by weight(e.g., at least 60%, 70%, 80%, 90%, 95%, or 98% by weight) of thewater-soluble, non-peptidic, and non-nucleotidic polymer backbone havingat least one terminus covalently bonded to a structure of formula (2).

In some embodiments, the method further can include (i) removing thehydrophobic separation handle(s) from the structure of formula (1) and(ii) optionally reacting the compound obtained in step (i) with anactivating agent before reacting with a biologically active molecule orthe derivative.

This document also features a conjugate, or a pharmaceuticallyacceptable salt thereof, including a structure of formula (10):

or a salt form thereof, wherein: polymer is a linear, water-soluble,non-peptidic, and non-nucleotidic polymer backbone, wherein each linkinggroup is bonded at a different terminus of the polymer; E and E¹ areindependently O or S; K and K₁ are independently selected from the groupconsisting of: alkylene, alkyleneoxyalkylene, and oligomericalkyleneoxyalkylene; G and G₁ are independently absent or are selectedfrom the group consisting of: alkoxy and a hydrophobic separationhandle; each pair of Z¹ and Z² and Z³ and Z⁴ are independently selectedfrom O and NH, wherein only one of each pair of Z¹ and Z² and Z³ and Z⁴can be NH; L and L¹ are independently selected from the group consistingof: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene,oligomeric alkyleneoxyalkylene, and unsubstituted and substitutedarylene; L² is a covalent linking moiety between L on the polymerbackbone and B; L³ is a covalent linking moiety between L on the polymerbackbone and B¹; and B and B¹ are independently a biologically activemolecule or a derivative of a biologically active molecule (e.g., a TNFinhibitor, a derivative of a TNF inhibitor, insulin, a derivative ofinsulin, omalizumab, a derivative of omalizumab, a clotting factor, aderivative of a clotting factor, a polypeptide that boosts red or whitecell production, a derivative of a polypeptide that boosts red or whitecell production, an antibody, or a derivative of an antibody), a drug, adetectable group, a separation moiety, wherein at least one of B and B¹is a biologically active molecule or a derivative of a biologicallyactive molecule. For example, B and B¹ each can be selected from thegroup consisting of: a TNF inhibitor, insulin, omalizumab, a clottingfactor, a polypeptide that boosts red or white cell production, and anantibody.

This document also features a method of making a biologically activemolecule conjugate. The method includes reacting a biologically activemolecule or a derivative thereof with a preparation that includes acompound of formula (3):

or a salt form thereof,

wherein: polymer is a linear, water-soluble, non-peptidic, andnon-nucleotidic polymer backbone, wherein M² and the phosphonate-derivedfunctional group are bonded at a different terminus of the polymer; Eand E¹ are independently O or S; K is selected from the group consistingof: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; Gis selected from the group consisting of: hydrogen, alkoxy, and ahydrophobic separation handle; Z¹ and Z² are independently selected fromO and NH, wherein only one of Z¹ and Z² can be NH; L is selected fromthe group consisting of: a divalent radical of nucleoside, alkylene,alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstitutedand substituted arylene; M is selected from a protected group that whendeprotected is reactive with a biologically active molecule or thederivative or a group reactive with a biologically active molecule orthe derivative; M² is selected from O, S or NH; and R is absent, aprotecting group, a hydrophobic separation handle, or an activatinggroup; R² is hydrogen or a protecting group; wherein when M is aprotected group that when deprotected is reactive with a biologicallyactive molecule or the derivative, then R is a protecting group or ahydrophobic separation handle; and wherein when M is a group reactivewith a biologically active molecule or the derivative, R is absent,hydrogen, or an activating group.

In some embodiments, one of Z¹ and Z² is NH and the other is O. In someembodiments, Z¹ is O and Z² is NH. In some embodiments, Z¹ is NH and Z²is O. In some embodiments, both Z¹ and Z² are O.

A group reactive with a biologically active molecule or the derivativecan be selected from the group consisting of: hydroxyl, amine, thiol,carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide,acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide,dithiopyridine, and iodoacetamide.

In some embodiments, K is selected from the group consisting of:methylene, ethylene, propylene, isopropylene, butylene, isobutylene,sec-butylene, tert-butylene, and hexylene, or a residue from diethyleneglycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol.

In some embodiments, G is a substituted or unsubstituted trityloxy. Forexample, G can be a monoalkoxy substituted trityloxy group or a dialkoxysubstituted trityloxy group.

In some embodiments, R² is absent or selected from the group consistingof trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,fluorenylmethyloxycarbonyl, alkylcarboxyl, benzoyl, tetrahydropyranyl,and methyl.

In some embodiments, the polymer is poly(ethylene glycol).

In some embodiments, the preparation includes at least 50% by weight(e.g., at least 60%, 70%, 80%, 90%, 95%, or 98% by weight) of thewater-soluble, non-peptidic, and non-nucleotidic polymer backbone havingat least one terminus covalently bonded to a structure of formula (3).

In some embodiments, the method further can include (i) removing thehydrophobic separation handle(s) from the structure of formula (1) and(ii) optionally reacting the compound obtained in step (i) with anactivating agent before reacting with a biologically active molecule orthe derivative.

In some embodiments, the method further can include removing thehydrophobic separation handle(s) from the structure of formula (1) afterreacting with a biologically active molecule or the derivative.

This document also features a conjugate, or a pharmaceuticallyacceptable salt thereof, that includes a compound of formula (11):

or a salt form thereof,

wherein: polymer is a linear, water-soluble, non-peptidic, andnon-nucleotidic polymer backbone, wherein M² and the phosphonate-derivedfunctional group are bonded at a different terminus of the polymer; Eand E¹ are independently O or S; K is selected from the group consistingof: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; Gis selected from the group consisting of: hydrogen, alkoxy, and ahydrophobic separation handle; Z¹ and Z² are independently selected fromO and NH, wherein only one of Z¹ and Z² can be NH; L is selected fromthe group consisting of: a divalent radical of nucleoside, alkylene,alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstitutedand substituted arylene; L² is a covalent linking moiety between L onthe polymer backbone and B; L⁴ is a covalent linking moiety between L onthe polymer backbone and B¹; and B and B¹ are independently abiologically active molecule or a derivative of a biologically activemolecule (e.g., a TNF inhibitor, a derivative of a TNF inhibitor,insulin, a derivative of insulin, omalizumab, a derivative ofomalizumab, a clotting factor, a derivative of a clotting factor, apolypeptide that boosts red or white cell production, a derivative of apolypeptide that boosts red or white cell production, an antibody, or aderivative of an antibody), a biologic other than the biologicallyactive molecule, a drug, a detectable group, a separation moiety,wherein at least one of B and B¹ is a biologically active molecule or aderivative of a biologically active molecule. For example, B and B¹ eachcan be selected from the group consisting of: a TNF inhibitor, insulin,omalizumab, a clotting factor, a polypeptide that boosts red or whitecell production, an antibody,

This document also features a method of preparing a compound thatincludes a water-soluble, non-peptidic, and non-nucleotidic polymerbackbone having at least one terminus covalently bonded to a structureof formula (9):

or a salt thereof,

wherein: A is the point of covalent bonding to the terminus of thepolymer backbone; E is O or S; Y represents an optionally substitutedresidue selected from alkyl, cycloalkyl, heterocyclyl, aryl, andheteroaryl; Z¹ and Z² are independently selected from O and NH, whereinonly one of Z¹ and Z² can be NH; L is selected from the group consistingof: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene,oligomeric alkyleneoxyalkylene, and unsubstituted and substitutedarylene; L2 is a covalent linking moiety between L on the polymerbackbone and B; and B is a biologically active molecule or a derivativethereof. The method includes

(a) providing a composition comprising a compound of formula (8):

wherein: A is the point of covalent bonding to the terminus of thepolymer backbone; E is O or S; Y represents an optionally substitutedresidue selected from alkyl, cycloalkyl, heterocyclyl, aryl, andheteroaryl; Z¹ and Z² are independently selected from O and NH, whereinonly one of Z¹ and Z² can be NH; L is selected from the group consistingof: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene,oligomeric alkyleneoxyalkylene, and unsubstituted and substitutedarylene; M is a protected group that when deprotected is reactive with abiologically active molecule or a derivative thereof. R is a hydrophobicseparation handle; R¹ is absent or a hydrophobic separation handle; and

(b) removing the hydrophobic separation handle(s);

(c) optionally reacting the compound obtained in step (b) with anactivating agent; and

(d) reacting the compound obtained in step (b), or, optionally in step(c), with a biologically active molecule or a derivative thereof.

In some embodiments, one of Z¹ and Z² is NH and the other is O. In someembodiments, Z¹ is O and Z² is NH. In some embodiments, Z¹ is NH and Z²is O. In some embodiments, both Z¹ and Z² are O.

The protected group M can be, when deprotected, selected from the groupconsisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal,dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone,hydrazide, aminoxy, maleimide, dithiopyridine, and iodoacetamide.

In some embodiments, R is selected from the group consisting of: trityl,monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.

In some embodiments, the polymer backbone is selected from the groupconsisting of poly(alkylene glycol), poly(oxyethylated polyol),poly(olefinic alcohol), poly(α-hydroxy acid), poly(vinyl alcohol),polyoxazoline, and copolymers. For example, the polymer backbone can bepoly(ethylene glycol).

In some embodiments, reaction step (d) is carried out in the presence ofwater or a protic solvent. In some embodiments, the compound of formula(8) is essentially pure.

In some embodiments, the compound of formula (8) comprises at least 98%by weight of the composition.

In one aspect, this document features a composition that includes any ofthe conjugates described herein and a pharmaceutically acceptableexcipient. The polymer backbone of the conjugate can be poly(ethyleneglycol).

In yet another aspect, this document features a method of treating apatient diagnosed with a disease selected from the group consisting of:an inflammatory disease; diabetes; asthma; hemophilia, factor VIIdeficiency, or other clotting disorder; The method includesadministering to the patient an effective amount of any of theconjugates described herein. The polymer backbone of the conjugate canbe poly(ethylene glycol).

This disclosure utilizes phosphoramidites as reagents interacting withpolymers to form a phosphotriester-type of linker between a polymer andlinking group. A similar process is used commonly in the chemistry ofnucleic acids. Formation of a phosphotriester bond, in the chemistry ofnucleic acids, is often followed by its partial hydrolysis(deprotection) to the phosphodiester bond, because phosphodiester bondsare naturally occurring. The present disclosure provides an uncommon andunnatural phosphotriester linkage which is enzymatically resistant,offering a stable linker between a polymer and a biologically activemolecule such as a TNF inhibitor, insulin, omalizumab, a clottingfactor, a polypeptide that boosts red or white cell production, anantibody, or a derivative thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SEQ ID NO. 1, the amino acid sequence omalizumab (XOLAIR™).

FIG. 2 provides a photograph of a SDS electrophoresis gel of the productof the conjugation of omalizumab to a PEG reagent as provided hereinstained with Coomassie blue.

FIG. 3 provides a photograph of a SDS electrophoresis gel of the productof the conjugation of omalizumab to a PEG reagent as provided hereinstained with barium chloride and iodine.

FIG. 4 provides a photograph of a SDS electrophoresis gel of the productof the conjugation of omalizumab to a PEG reagent as provided hereinstained with Coomassie blue.

FIG. 5 provides a photograph of a SDS electrophoresis gel of the productof the conjugation of omalizumab to a PEG reagent as provided hereinstained with Coomassie blue.

FIG. 6 shows HPLC Gel Filtration analysis of free omalizumab andreaction mixture obtained after pegylation of omalizumab.

FIG. 7 shows SEQ ID NO. 2, the amino acid sequence of insulin.

FIG. 8 illustrates the HPLC Gel Filtration chromatogram of lysozyme(dashed line) and the lysozyme pegylation reaction product (solid line).

FIG. 9 illustrates the HPLC Gel Filtration chromatogram of insulin(dashed line) and insulin pegylation reaction mixture (solid line).

FIG. 10 shows SEQ ID NO. 3, the amino acid sequence etanercept(ENBREL®).

FIG. 11 provides a photograph of a SDS electrophoresis gel of theproduct of the conjugation of entanercept to a PEG reagent as providedherein stained with Coomassie blue.

FIG. 12 illustrates the HPLC Gel Filtration chromatogram of etanercept(dashed line) and the etanercept pegylation reaction product (solidline).

FIG. 13 shows rhTNF-alpha concentrations following over-night incubationat 4-8° C. with various concentrations of etanercept (non-PEGylated),PEG20-etanercept (5 eq), or PEG20-etanercept (10 eq). A fixedconcentration of 2.9 ng/ml rhTNF-alpha was used in all incubations.PEGylation was conducted in 5 time molar excess (5 eq) or 10 time molarexcess (10 eq) of PEG20 compared etanercept. IC₅₀ is expressed as grametanercept per ml.

FIG. 14 illustrates the dissociation over time of ¹²⁵I-omalizumab boundto human IgE with or without unlabeled omalizumab or PEGylatedomalizumab.

DETAILED DESCRIPTION

This document provides conjugates of a biologically active molecule(e.g., a TNF inhibitor, insulin, omalizumab, a clotting factor, apolypeptide that boosts red or white cell production, or an antibody) ora derivative thereof and functionalized (e.g., mono- or bi-functional)polymers (e.g., polyethylene glycol and related polymers) as well asmethods and materials for making and using such conjugates.

Non-limiting examples of a biologically active molecule include a TNFinhibitor, insulin, omalizumab, a clotting factor, a polypeptide thatboosts red or white cell production, and an antibody.

As used herein, the term “TNF inhibitor” includes antibodies or fusionproteins that bind to TNF alpha. Non-limiting examples of TNF inhibitorsinclude etanercept (Enbrel®, sold by Amgen and Pfizer); infliximab(Remicade®, sold by Janssen Biotech, Inc.); adalimumab (Humira®, sold byAbbott Laboratories); certolizumab pegol (Cimzia®); and Golimumab(Simponi®, sold by Janssen Biotech, Inc.).

Etanercept (Enbrel®) is a fusion protein of human soluble TNF receptor 2to the Fc component of human IgG₁. It is a TNF inhibitor that binds toTNF alpha and is used to treat inflammatory diseases e.g., rheumatoidarthritis, plaque psoriasis, psoriatic arthritis, juvenile idiopathicarthritis (JIA), and ankylosing spondylitis (AS)).

Infliximab (Remicade®) is a chimeric mouse-human monoclonal antibodythat specifically binds TNF alpha. It is used for the treatment ofpsoriasis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis,rheumatoid arthritis, and ulcerative colitis.

Adalimumab (Humira®) is a fully human monoclonal antibody that binds TNFalpha and is used for the treatment of rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, moderate to severechronic psoriasis, and juvenile idiopathic arthritis.

Certolizumab pegol (Cimzia®) is a pegylated fragment Fab′ of humanizedTNF inhibitor monoclonal antibody.

Golimumab (Simponi®) is a human monoclonal antibody that targets TNFalpha, and is used to treat severely active rheumatoid arthritis,psoriatic arthritis, and ankylosing spondylitis.

As used herein, the term “insulin” includes human and other mammalianinsulin polypeptides as well as insulin analogs that have one or moreamino acid substitutions. Human insulin is composed of A and Bpolypeptide chains bound together by disulfide bonds, where the A chainis 21 amino acids in length and the B chain is 30 amino acids in length,51 amino acids in total. Insulin and analogs can be recombinantlyproduced (e.g., in a non-pathogenic organism). Non-limiting examples ofsuitable insulin analogs include insulin lispro (HUMALOG, Eli Lilly),insulin aspart (NovoLog/NovoRapid, Novo Nordisk), insulin detemir(Levemir®, Novo Nordisk), Insulin glargine (Lantus®, Sanofi-Aventis),and insulin glulisine (Apidra®, Sanofi-Aventis). See Werner andChantelau, Diabetol Metab Syndr. 3: 13 (2011) for a comparison of thebioactivity of human insulin and insulin analogs.

Insulin lispro is a rapid-acting human insulin analog used to lowerblood glucose levels, and contains a substitution of lysine for prolineat position B28, and a substitution of proline for lysine at positionB29.

Insulin aspart is a rapid-acting human insulin analog used to lowerblood glucose levels, and contains a substitution of an aspartic acidfor proline at position B28.

Insulin detemir is a long-acting insulin analog used for maintaining thebasal level of insulin. Insulin determir contains myristic acid is boundto the lysine at position B29.

Insulin glargine is a long-acting insulin analog used for lower bloodglucose, and contains a substitution of a glycine for asparagine atposition A21 and contains two arginines added to the C-terminus of theB-chain.

Insulin glulisine is a rapid acting human insulin analog used to lowerblood glucose levels, and contains a substitution of a lysine forasparagine at position B3 and the substitution of a glutamic acid forlysine at position B29.

Omalizumab (Xolair®, Novartis Pharmaceuticals, East Hanover, N.J.;Genentech Inc., South San Francisco, Calif.) is a recombinant humanizedmonoclonal antibody (rhuMAb-E25) for treatment of moderate to severepersistent asthma. Omalizumab inhibits the binding of IgE to thehigh-affinity IgE receptor FcεRI by binding to an epitope on IgE thatoverlaps with the site to which FcεRI binds. Consequently, circulatingfree IgE is reduced, IgE is prevented from attaching to mast cells andbasophils, and FcεRI receptor expression is down-regulated. See, e.g.,Sandstrom, J Asthma Allergy, 2: 49-62 (2009).

As used herein, the term “clotting factor” includes human or othermammalian clotting factors such as factor VII and activated factor VII(factor VIIa), factor VIII and activated factor VIII (factor VIIIa),factor IX and activated factor IX (factor IXa), antithrombin, humanfibrinogen/human thrombin, and protein C and activated protein C (APC).Such clotting factors can be recombinantly produced.

Factor VII functions in the initial stage of blood clotting and is a keyelement in forming blood clots. The inactive precursor has low enzymeactivity that is increased by proteolytic cleavage to form factor VIIa.This activation can be catalyzed by factor Xa as well as by VIIa-tissuefactor, an integral membrane protein found in a number of cell types.See Fiore, et al., J. Biol. Chem., 269:143-149 (1994). Factor VIIa canactivate blood clotting factors IX and X. Eptacog alfa (activated) (soldby Novo Nordisk) can be used to replace missing factor VII in patientswith factor VII deficiency and also can be used in haemophilia patientswho have developed inhibitors to factor VIII or IX.

Factor VIII is activated by thrombin (Factor IIa), and then can interactwith Factor IXa in the coagulation cascade. It is a cofactor to FactorIXa in the activation of Factor X, which, in turn, with its cofactorFactor Va, activates more thrombin. Thrombin cleaves fibrinogen intofibrin which polymerizes and crosslinks (using Factor XIII) into a bloodclot. Factor VIII deficiency results in hemophilia A and Factor IXdeficiency causes hemophilia B. Moroctocog alfa (glycoprotein (tradename ReFacto®, Xyntha®) are commercially available for Factor VIIIreplacement therapy. Factor VIII replacement therapy is limited due todevelopment of high-titer inhibitory factor VIII antibodies in somepatients. Nonacog alfa (Benefix®) is a human recombinant factor IXproduct used in treating hemophilia B patients.

Antithrombin is a small glycoprotein that inactivates several enzymes ofcoagulation system. Its activity enhanced by heparin. Antithrombin alfa(e.g., ATryn®) is commercially available.

Protein C is activated by thrombin in the presence of thrombomodulin, anintegral membrane protein of endothelial cells. Esmon, et al., J. Biol.Chem., 257:859-864 (1982). Activated protein C (APC) degrades factors Vaand VIIIa in combination with its cofactor, protein S. Resistance to APCis the most common form of inherited thrombosis disease. Dahlback,Blood, 85:607-614 (1995).

As used herein, the term “polypeptide that boosts red blood cellproduction” includes human erythropoetin (e.g., epoetin alpha (Epogen®,Amgen), epoetin beta (NeoRecormon®, Roche), epoetin theta (Eporatio®),or epoetin zeta) and other mammalian erythropoetin polypeptides as wellas analogs that have one or more amino acid substitutions (e.g.,darbepoetin alpha (Aranesp®, Amgen)) or other chemical modifications. Asused herein, the term “polypeptide that boosts white blood cellproduction” includes granulocyte colony-stimulating factor (G-CSF)analogs such as filgrastim (e.g., Neupogen®), which is identical tohuman G-CSF except for the addition of an N-terminal methioninenecessary for expression in E coli. Figrastim is nonglycosylated. Suchpolypeptides can be recombinantly produced.

Epoetin alpha, beta, theta, and zeta are structurally similar and havethe same polypeptide receptor binding sites. However, there are subtledifferences in the glycosylation patterns of different types ofepoetins. See Sergei et al., Arch Drug Inf., 4(3): 33-41 (2011); andStorring et al., Br J Haematol. 100(1):79-89 (1998). Darbepoetin is a165-amino acid protein that differs from recombinant humanerythropoietin in containing 5 N-linked oligosaccharide chains, whereasrecombinant human erythropoietin contains 3 chains. The two additionalN-glycosylation sites result from amino acid substitutions in theerythropoietin peptide backbone. The additional carbohydrate chainsincrease the approximate molecular weight of the glycoprotein from30,000 to 37,000 daltons.

Non-limiting examples of antibodies that can be conjugated to afunctionalized polymer include, abatacept (Orencia®), alemtuzumab(marketed as Campath, MabCampath or Campath-1H); basiliximab(Simulect®), belimumab (Benlysta®), besilesomab (Scintimun®),bevacizumab (Avastin®), canakinumab (Ilaris®), catumaxomab (Removab®),cetuximab (Erbitux®), denosumab (Prolia®, Xgeva®), eculizumab(Soliris®), ipilimumab (also known as MDX-010 or MDX-101, Yervoy®),natalizumab (Tysabri®), ofatumumab (Arzerra®), palivizumab (Synagis®),panitumumab (Vectibix®), ranibizumab (Lucentis®), rituximab (Rituxan®,MabThera®), tocilizumab (Actemra®, RoActemra®), trastuzumab(Herceptin®), and ustekinumab (Stelara®).

The antibody also can be an antibody drug conjugate (ADC). An ADC caninclude, for example, an antibody such as a monoclonal antibody, orantibody fragment such as a single-chain variable fragment, linked to adrug (e.g., a cytotoxic drug). In some embodiments, the antibody orfragment and drug can be linked via a cleavable linker (e.g., adisulfide-based linker or peptide linker). In some embodiments, theantibody or fragment and drug can be linked via a noncleavable linker.

For example, this document provides an antibody or a derivative thereofsuch as abatacept (Orencia®), alemtuzumab (marketed as Campath,MabCampath or Campath-1H); basiliximab (Simulect®), belimumab(Benlysta®), besilesomab (Scintimun®), bevacizumab (Avastin®),canakinumab (Ilaris®), catumaxomab (Removab®), cetuximab (Erbitux®),denosumab (Prolia®, Xgeva®), eculizumab (Soliris®), ipilimumab (alsoknown as MDX-010 or MDX-101, Yervoy®), natalizumab (Tysabri®),ofatumumab (Arzerra®), palivizumab (Synagis®), panitumumab (Vectibix®),ranibizumab (Lucentis®), rituximab (Rituxan®, MabThera®), tocilizumab(Actemra®, RoActemra®), trastuzumab (Herceptin®), or ustekinumab(Stelara®) conjugated to functionalized PEG polymers.

In some embodiments, a biologically active molecule can be agalsidasealfa (Replagal®), agalsidase beta (Fabrazyme®), alglucosidase alfa(Myozyme®), anakinra (Kineret®), bortezomib (Velcade®), conestat alfa(Ruconest®), choriogonadotropin alfa (e.g., Ovidrel®), cinacalcet(Sensipar®), Corifollitropin alfa (Elonva®), dibotermin alfa(InductOs®), eltrombopag (Promacta®), eptotermin alfa (Osigraft®),erlotinib (Tarceva®), follitropin alfa/lutropin alfa, follitropin beta,galsulfase (Naglazyme®), Gefitinib (Iressa®), Human normalimmunoglobulin, human normal immunoglobulin (ivig), human normalimmunoglobulin (SCIg), idursulfase (Elaprase®), Imiglucerase(Cerezyme®), interferon alfa-2b, interferon beta-la, interferon beta-1b,Lapatinib (Tykerb®, Tyverb®), Laronidase (Aldurazyme®), Pazopanib(Votrient), pegaptanib, pegfilgrastim, peginterferon alfa-2b,Rasburicase (Elitek®), Reteplase (Retavase®), Somatropin, Sorafenib(Nexavar®), Tenecteplase (TNKase), thyrotropin alfa, Vandetanib(Caprelsa®), or Vemurafenib (Zelboraf®).

The conjugates described herein can have an altered pharmacokinetic andpharmacodynamic profile, including, for example, one or more of reduceddosage frequency, extended circulation time, and reduced antigenicproperties. As described herein, a biologically active molecule or aderivative thereof can be is conjugated to a functionalized polymer thatincludes one or more linking groups selected from a phosphotriester, aphosphoramidate, a thiophosphotriester, and a thiophosphoramidate.Suitable functionalized polymers can include different linking groups ateach of its termini. A suitable functionalized polymer also can bemodified at at least one terminus with a blocking group (e.g., methoxygroup) and functionalized at at least another terminus with a linkinggroup as described herein.

For example, this document provides a biologically active molecule or aderivative thereof conjugated to functionalized PEG polymers. In somecases, a functionalized PEG polymer has one terminus containing afunctional group covalently bound via a phosphotriester, aphosphoramidate, a thiophosphotriester, and a thiophosphoramidatelinking group.

In some cases, a PEG polymer is a linear PEG polymer (i.e., having twotermini). A linear PEG polymer can be functionalized as described hereinat one or both termini with the same or different functional grouplinked via the same or different linking groups. In some cases, one ofthe termini of a linear PEG polymer is blocked with a blocking group(e.g., methoxy or a protecting group) and the other termini isfunctionalized with a functional group linked as described herein. Insome cases, one of the termini of a linear PEG polymer is functionalizedwith a phosphotriester linking group and the other termini isfunctionalized with a phosphoramidate linking group. In other cases,both termini are functionalized with the same or differentphosphotriester linking groups. In yet other cases, both termini arefunctionalized with the same or different phosphoramidate linkinggroups.

Definitions

For the terms “for example” and “such as,” and grammatical equivalencesthereof, the phrase “and without limitation” is understood to followunless explicitly stated otherwise. As used herein, the term “about” ismeant to account for variations due to experimental error. Allmeasurements reported herein are understood to be modified by the term“about”, whether or not the term is explicitly used, unless explicitlystated otherwise. As used herein, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise.

The term “alkyl” includes straight-chain alkyl groups (e.g., methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.)and branched-chain alkyl groups (e.g., isopropyl, tert-butyl, isobutyl,etc.). In certain embodiments, a straight chain or branched chain alkylhas twelve or fewer carbon atoms in its backbone (e.g., C₁₋₁₂ forstraight chain; C₃₋₁₂ for branched chain). The term C₁₋₁₂ includes alkylgroups containing 1 to 12 carbon atoms.

The term “alkenyl” includes aliphatic groups that may or may not besubstituted, as described above for alkyls, containing at least onedouble bond and at least two carbon atoms. For example, the term“alkenyl” includes straight-chain alkenyl groups (e.g., ethenyl,propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, anddecenyl) and branched-chain alkenyl groups. In certain embodiments, astraight chain or branched chain alkenyl group has twelve or fewercarbon atoms in its backbone (e.g., C₂₋₁₂ for straight chain; C₃₋₁₂ forbranched chain). The term C₂₋₁₂ includes alkenyl groups containing 2 to12 carbon atoms.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond and two carbon atoms. Forexample, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, and decynyl) and branched-chain alkynyl groups. In certainembodiments, a straight chain or branched chain alkynyl group has twelveor fewer carbon atoms in its backbone (e.g., C₂₋₁₂ for straight chain;C₃₋₁₂ for branched chain). The term C₂₋₆ includes alkynyl groupscontaining 2 to 12 carbon atoms.

The term “alkylene” by itself or as part of another molecule means adivalent radical derived from a linear or branched alkane, asexemplified by (—CH₂—)_(n), wherein n may be 1 to 24 (e.g., 1 to 20, 1to 18, 1 to 16, 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to4, 1 to 3, 1 to 2, 2 to 24, 2 to 12, 2 to 8). By way of example only,such groups include, but are not limited to, groups having 10 or fewercarbon atoms such as the structures —CH₂CH₂— and CH₂CH₂CH₂CH₂—. A “loweralkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The term “alkoxy” is used in its conventional sense, and refers to alkylgroups linked to molecules via an oxygen atom. In some embodiments, analkoxy has twelve or fewer carbon atoms in its backbone (e.g., a C₁₋₁₂alkoxy). For example, C₁₋₁₀, C₁₋₈, C₁₋₆, C₁₋₄, C₁₋₃, or C₁₋₂.Non-limiting examples of an alkoxy group include methoxy, ethoxy,propoxy, butoxy, and hexoxy.

The term “alkyleneoxyalkylene,” as used herein, refers to a divalentradical derived from a linear or branched alkyloxyalkane, asexemplified, but not limited by, —CH₂—CH₂—O—CH₂—CH₂— and —CH₂—CH₂—O—. Byway of example only, such groups include, but are not limited to, groupshaving the formula —(CH₂)_(n)—O—(CH₂)_(m)—, wherein n is an integer from1 to 50 (e.g., 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1to 12, 1 to 10, 1 to 6, 1 to 6, 1 to 2, 1 to 3, 1 to 4, 2 to 50, 5 to50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to18) and m is an integer from 0 to 50 (e.g., 1 to 40, 1 to 30, 1 to 25, 1to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to 2, 1 to 3, 1 to4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20to 30, and 6 to 18).

The term “oligomeric alkyleneoxyalkylene” refers to p-repetitivealkyleneoxyalkylene wherein p is an integer of between 2 and 24 (e.g., 2to 20, 2 to 18, 2 to 16, 2 to 15, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to5, 2 to 4, 2 to 3). By way of example only, such groups include, but arenot limited to, groups having the formula —((CH₂)_(n)—O—(CH₂)_(m))_(p)—,wherein n is an integer from 1 to 50 (e.g., 1 to 40, 1 to 30, 1 to 25, 1to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to 6, 1 to 2, 1 to3, 1 to 4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to12, 20 to 30, and 6 to 18), m is an integer from 0 to 50 (e.g., 1 to 40,1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1to 2, 1 to 3, 1 to 4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5to 15, 2 to 12, 20 to 30, and 6 to 18), and each p is independently aninteger from 1 to 10 (e.g., 1 to 8, 1 to 6, 1 to 5, 1 to 3, 2 to 10, 4to 10, 6 to 10, 2 to 8, and 3 to 6).

In general, the term “arylene” by itself or as part of another moleculemeans a divalent radical derived from an aryl, including, for example,5- and 6-membered single-ring aromatic groups, such as benzene andphenyl. Furthermore, the term “arylene” includes a divalent radicalderived from a multicyclic aryl group, e.g., tricyclic, bicyclic, suchas naphthalene and anthracene.

The term “substituted” means that an atom or group of atoms replaceshydrogen as a “substituent” attached to another group. For aryl andheteroaryl groups, the term “substituted”, unless otherwise indicated,refers to any level of substitution, namely mono, di, tri, tetra, orpenta substitution, where such substitution is permitted. Thesubstituents are independently selected, and substitution may be at anychemically accessible position. In some cases, two sites of substitutionmay come together to form a 3-10 membered cycloalkyl or heterocycloalkylring.

Substituents include, but are not limited to, halo, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₅-C₁₂ aralkyl, C₃-C₁₂cycloalkyl, C₄-C₁₂ cycloalkenyl, phenyl, substituted phenyl, toluoyl,xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₅-C₁₂ alkoxyaryl, C₅-C₁₂aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀ alkylsulfonyl,—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8, aryl, substitutedaryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substitutedheterocyclic radical, nitroalkyl, —NO₂, —CN, —NR⁹C(O)—(C₁-C₁₀ alkyl),—C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkthioalkyl, —C(O)O—(C₁-C₁₀ alkyl), —OH,SO₂, ═S, —COOH, —NR⁹ ₂, carbonyl, —C(O)—(C₁-C₁₀ alkyl)-CF₃, —C(O)—CF₃,C(O)NR⁹ ₂, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀ aryl), —C(O)—(C₆-C₁₀ aryl),—(CH₂)_(m)—O—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein each m is from 1 to 8,—C(O)NR⁹ ₂, —C(S)NR⁹ ₂, —SO₂NR⁹ ₂, NR⁹C(O)NR⁹ ₂, —NR⁹C(S)NR⁹ ₂, saltsthereof, and the like. Each R⁹ group in the preceding list independentlyincludes, but is not limited to, H, alkyl or substituted alkyl, aryl orsubstituted aryl, or alkylaryl. Where substituent groups are specifiedby their conventional chemical formulas, written from left to right,they equally encompass the chemically identical substituents that wouldresult from writing the structure from right to left, for example,—CH₂O— is equivalent to —OCH₂—.

The term “polymer backbone” refers to the main chain of a linear orbranched polymer.

The term “water-soluble polymer backbone” refers to a polymer backbonehaving water-solubility or dispersibility in water at ambienttemperatures and a pH of about 7. For instance, a polyethylene glycolbackbone is considered to be water-soluble if the correspondingpolyethylene glycol can be solubilized or dispersed in water at ambienttemperatures and a pH of about 7.

The term “nucleotidic polymer” refers to a single- or double-strandedpolymer chain composed of two or more nucleic acids. The term “nucleicacid” refers to deoxyribonucleotides or ribonucleotides. By way ofexample only, such nucleic acids and nucleic acid polymers include, butare not limited to, (i) analogues of natural nucleotides which havesimilar binding properties as a reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides; (ii)oligonucleotide analogs including, but are not limited to, PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidites, and the like); (iii)conservatively modified variants thereof (including but not limited to,degenerate codon substitutions) and complementary sequences.

The term “peptidic polymer” refers to a polymer of two or more aminoacid residues. The term applies to naturally occurring amino acidpolymers as well as amino acid polymers in which one or more amino acidresidues is a non-natural amino acid.

As used herein, a “biologically active molecule” includes any moleculewhich can have a biological effect. Examples of biologically activemolecules include therapeutic agents, small molecules, oligo- andpolypeptides, oligonucleotides, coding DNA sequences, antisense DNAsequences, mRNAs, antisense RNA sequences, RNAis, and siRNAs,carbohydrates, lipids, growth factors, enzymes, transcription factors,toxins, antigenic peptides (as for vaccines), antibodies, and antibodyfragments.

The term “group reactive with a biologically active molecule” refers toa functional group that can be covalently bound to a functional group ofa biologically active molecule.

The terms “protecting group” and “protective group” refer to a moietythat reversibly chemically modifies a functional group in order toobtain chemoselectivity or in order to reduce degradation in one or moresubsequent chemical reactions. Suitable protecting groups are well knownin the art (see, e.g., Greene and Wuts, Protective Groups in OrganicSynthesis, 3rd Ed., John Wiley & Sons, New York, N.Y., 1999, which isincorporated herein by reference in its entirety).

The term “detectable functional group” refers a functional group thatphysically or chemically interacts with its environment to produce asignal or product that can be detected by analytical and/or imagingmethods such as visible, UV-, IR-, NIR-light, X-Ray, and NMR-basedimaging methods, enzymatic assays, and UV-, IR-, NMR-, X-ray-, and massspectrometry-based analytics.

As used herein, a “fluorophore” is a chemical group that can be excitedby light to emit fluorescence. Some suitable fluorophores may be excitedby light to emit phosphorescence. As used herein, a “dye” may include afluorophore. Non-limiting examples of a fluorophore include: 1,5IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein);5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX(carboxy-X-rhodamine); 5-TAMIA (5-Carboxytetranethylrhodamine);6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin;7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin;9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA(9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red;Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Alexa Fluor 350™;Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™;Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™;Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red;Allophycocyanin (APC); AMC; AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X;Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); AnilinBlue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTS; AstrazonBrilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G;Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); Berberine Sulphate;Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein;BFP/GFP FRET; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); BlancophorFFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy 492/515; Bodipy 493/503;Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy FL; Bodipy FL ATP;Bodipy F1-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate;Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1;BO-PRO™-3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; CalciumCrimson™; Calcium Green; Calcium Orange; Calcofluor White;Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow;Catecholamine; CCF2 (GeneBlazer); CFDA; CFP-Cyan Fluorescent Protein;CFP/YFP FRET; Chlorophyll; Chromomycin A; CL-NERF (Ratio Dye, pH);CMFDA; Coelenterazine f; Coelenterazine fcp; Coelenterazine h;Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; CoelenterazineO; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTCFormazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; CyanGFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine;Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (DichlorodihydrofluoresceinDiacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS(non-ratio); DiA (4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate(DCFH); DiD-Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydorhodamine123 (DHR); DiI (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR(DiIC18(7)); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP;ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide;Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III)chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; FlazoOrange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate;Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX;FM 1-43™; FM 4-46; Fura Red™; Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF;Genacryl Brilliant Red B; Genacryl Brilliant Yellow 1OGF; Genacryl Pink3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T); GFP red shifted(rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, WVexcitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue;Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS;Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine;Indo-1; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); IntrawhiteCf; JC-1; JO-JO-1; JO-PRO-1; Laurodan; LDS 751 (DNA); LDS 751 (RNA);Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine;Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1;Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso TrackerGreen; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue;LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red(Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; MagnesiumGreen; Magnesium Orange; Malachite Green; Marina Blue; Maxilon BrilliantFlavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin;Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; MitotrackerRed; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH);Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine;Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; NuclearYellow; Nylosan Brilliant Iavin E8G; Oregon Green; Oregon Green 488-X;Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514;Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP;PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); PhorwiteAR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613[PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110;Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green;Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; RhodamineWT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); RsGFP; S65A;S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange;Sevron Yellow L; sgBFP™; sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (superglow GFP); SITS; SITS (Primuline); SITS (Stilbene IsothiosulphonicAcid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARFI; SodiumGreen; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ(6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange;Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange;Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange;Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5;TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; UranineB; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F;Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. Asused herein, a “fluorophore” may include a salt of the fluorophore.

Fluorophores may include derivatives that have been modified tofacilitate conjugation to another reactive molecule. As such,fluorophores may include amine-reactive derivatives such asisothiocyanate derivatives and/or succinimidyl ester derivatives of thefluorophore.

The term “hydrophobic separation handle” refers to a moiety that whenattached to or part of a compound reduces the hydrophilicity of thatcompound, i.e. reduces its tendency to be solved or dispersed in water.The term “separation handle” refers to a moiety that when attached to orpart of a compound alters the mobility of that compound in achromatographic method such that its separation from contaminants isimproved. Alternatively, the term “separation handle” refers to a moietythat when attached to or part of a compound improves the yield of thecompound in a chromatographic method in comparison to a hydrogensubstituent.

The term “hydrophobicity” refers to the relative degree with which acompound or moiety is solved or dispersed in a non-aqueous solvent suchas n-octanol. The degree of hydrophobicity or hydrophilicity of acompound or moiety can be measured using methods known in the art, suchas, reversed phase chromatography and other chromatographic methods,partitioning, accessible surface area methods, and measurement ofphysical properties such as partial molar heat capacity, transitiontemperature and surface tension.

The term “activating group” refers to a moiety that increases thecapability of the group reactive with a biologically active molecule toform a covalent bond with a biologically active molecule. Usually thesegroups increase or decrease the electronegativity of a selected moietyso it becomes more nucleophilic or more electrophilic. Non-limitingexamples of an activating group include: lower alkylamino,diloweralkylamino, amino, halo, aryl, lower alkoxy, lower aralkoxy,aryloxy, mercapto, lower alkylthio, nitro, monophaloalkyl, dihaloalkyl,trihaloalkyl (e.g., CF₃), halo, formyl, lower alkanoyl, loweralkylsulfonyl, lower alkylsulfinyl, and the like.

The term “essentially pure” refers to chemical purity of a compoundprovided herein that may be substantially or essentially free of othercomponents which normally accompany or interact with the compound priorto purification. By way of example only, a compound may be “essentiallypure” when the preparation of the compound contains less than about 30%,less than about 25%, less than about 20%, less than about 15%, less thanabout 10%, less than about 5%, less than about 4%, less than about 3%,less than about 2%, or less than about 1% (by dry weight) ofcontaminating components. Thus, an “essentially pure” compound may havea purity level of about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 96%, about 97%, about 98%, about 99% or greater. Forthe purposes of this document, preparations of functionalized polymersor conjugates differing only in the length of their polymer chain areconsidered to be essentially pure. By way of example a preparation of amono-functionalized compound may be “essentially pure” when thepreparation contains less than about 30%, less than about 25%, less thanabout 20%, less than about 15%, less than about 10%, less than about 5%,less than about 4%, less than about 3%, less than about 2%, or less thanabout 1% (by dry weight) of contaminating unfunctionalized and/orpoly-functionalized polymers. An essentially pure compound may beobtained using chromatographic purification methods.

The term “protic solvent” is used herein to refer to solvents whichcomprise dissociable hydrogen ions. Examples of protic solvents includealcohols, such as ethanol, and methanol.

The phrase “pharmaceutically acceptable” is used herein to refer tothose inhibitors, materials, compositions, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

Some of the compounds provided herein are acidic and may form a saltwith a pharmaceutically acceptable cation. Some of the compounds hereincan be basic and accordingly, may form a salt with a pharmaceuticallyacceptable anion. All such salts, including di-salts are within thescope of the compositions described herein and they can be prepared byconventional methods. For example, salts can be prepared by contactingthe acidic and basic entities, in either an aqueous, non-aqueous orpartially aqueous medium. The salts are recovered by using at least oneof the following techniques: filtration, precipitation with anon-solvent followed by filtration, evaporation of the solvent, or, inthe case of aqueous solutions, lyophilization.

Salts, for example, include: (1) acid addition salts, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like; or formed with organicacids such as acetic acid, propionic acid, hexanoic acid,cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid,malonic acid, succinic acid, malic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoicacid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonicacid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,benzenesulfonic acid, 2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; and (2) salts formedwhen an acidic proton present in the parent compound either is replacedby a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base. Acceptable organicbases include ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine, and the like. Acceptable inorganicbases include aluminum hydroxide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like.

Compounds

The following compounds are suitable for conjugating to a biologicallyactive molecule or a derivative thereof.

Compounds of Formula (1)

Provided herein is a compound comprising a water-soluble,non-nucleotidic and non-peptidic polymer backbone having at least oneterminus covalently bonded to a structure of formula (1):

or pharmaceutical salt thereof wherein: A is the point of bonding to aterminus of the polymer backbone, E is an oxygen or sulfur atom, K isselected from the group consisting of alkylene, alkyleneoxyalkylene, oran oligomeric form of alkyleneoxyalkylene, G is selected from the groupconsisting of hydrogen, an alkoxy, or a hydrophobic separation handle,Z¹ and Z² can be oxygen or nitrogen, in such way that both Z¹ and Z² maybe oxygen, but when Z¹ is NH then Z² is oxygen, and when Z² is NH thenZ¹ is oxygen, L is selected from the group consisting of a divalentradical of a nucleoside, linear alkylene, branched alkylene,alkyleneoxyalkylene, oligomeric form of alkyleneoxyalkylene, arylene,and substituted arylene, M is a protected group that when deprotected isreactive with a biologically active molecule or a derivative thereof, agroup reactive with a biologically active molecule or a derivativethereof, or detectable functional group, R is a protecting group,activating group, hydrogen or absent.

A polymer backbone, as provided herein, can be branched or linear. Forexample, a polymer backbone can have from 2 to 100 termini (e.g., 2 to80, 2 to 75, 2 to 60, 2 to 50, 2 to 40, 2 to 35, 2 to 25, 2 to 10, 2 to5, 4 to 20, 5 to 25, 10 to 50, 25 to 75, 3 to 6, 5 to 15 termini). Insome embodiments, a polymer is linear and therefore has 2 termini. Insome embodiments, only one termini of a polymer backbone is covalentlybonded to the structure of formula (1). In some embodiments, wherein apolymer has two termini, both termini of the polymer backbone arecovalently bonded to the structure of formula (1).

A polymer backbone can be, for example, poly(alkylene glycol),poly(oxyethylated polyol), poly(olefinic alcohol), poly(α-hydroxy acid),poly(vinyl alcohol), polyoxazoline, or a copolymer thereof. Apolyalkylene glycol includes linear or branched polymeric polyetherpolyols. Such polyalkylene glycols, include, but are not limited to,polyethylene glycol, polypropylene glycol, polybutylene glycol, andderivatives thereof. Other exemplary embodiments are listed, forexample, in commercial supplier catalogs, such as ShearwaterCorporation's catalog “Polyethylene Glycol and Derivatives forBiomedical Applications” (2001). By way of example only, such polymericpolyether polyols have average molecular weights between about 0.1 kDato about 100 kDa. By way of example, such polymeric polyether polyolsinclude, but are not limited to, between about 500 Da and about 100,000Da or more. The molecular weight of the polymer may be between about 500Da and about 100,000 Da. For example, a polymer used herein can have amolecular weight of about 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da,80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da,15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da,4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da,and 500 Da. In some embodiments, the molecular weight of the polymer isbetween about 500 Da and about 50,000 Da. In some embodiments, themolecular weight of the polymer is between about 500 Da and about 40,000Da. In some embodiments, the molecular weight of the polymer is betweenabout 1,000 Da and about 40,000 Da. In some embodiments, the molecularweight of the polymer is between about 5,000 Da and about 40,000 Da. Insome embodiments, the molecular weight of the polymer is between about10,000 Da and about 40,000 Da.

In some embodiments, a polymer backbone is a linear or branchedpoly(ethylene glycol).

In some embodiments, the poly(ethylene glycol) molecule is a linearpolymer. The molecular weight of the linear chain PEG may be betweenabout 1,000 Da and about 100,000 Da. For example, a linear chain PEGused herein can have a molecular weight of about 100,000 Da, 95,000 Da,90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da,25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. Insome embodiments, the molecular weight of the linear chain PEG isbetween about 1,000 Da and about 50,000 Da. In some embodiments, themolecular weight of the linear chain PEG is between about 1,000 Da andabout 40,000 Da. In some embodiments, the molecular weight of the linearchain PEG is between about 5,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the linear chain PEG is betweenabout 5,000 Da and about 20,000 Da.

In some embodiments, the poly(ethylene glycol) molecule is a branchedpolymer. The molecular weight of the branched chain PEG may be betweenabout 1,000 Da and about 100,000 Da. For example, a branched chain PEGused herein can have a molecular weight of about 100,000 Da, 95,000 Da,90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da,25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. Insome embodiments, the molecular weight of the branched chain PEG isbetween about 1,000 Da and about 50,000 Da. In some embodiments, themolecular weight of the branched chain PEG is between about 1,000 Da andabout 40,000 Da. In some embodiments, the molecular weight of thebranched chain PEG is between about 5,000 Da and about 40,000 Da. Insome embodiments, the molecular weight of the branched chain PEG isbetween about 5,000 Da and about 20,000 Da.

In some embodiments, E is oxygen. In some embodiments, E is sulfur.

In some embodiments, K is a linear or branched alkylene. For example, Kcan be selected from the group consisting of: methylene, ethylene,propylene, isopropylene, butylene, isobutylene, sec-butylene,tert-butylene, and hexylene. In some embodiments, K can be analkyleneoxyalkylene or an oligomeric alkyleneoxyalkylene. For example, Kcan be a residue from diethylene glycol, triethylene glycol,tetraethylene glycol, or hexaethylene glycol. In some embodiments, K isselected from the group consisting of —(CH₂)_(n)— and—((CH₂)_(n)—O—(CH₂)_(m))_(p)—, wherein n is an integer from 1 to 50(e.g., 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1to 10, 1 to 6, 1 to 6, 1 to 2, 1 to 3, 1 to 4, 2 to 50, 5 to 50, 10 to50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), m isan integer from 0 to 50 (e.g., 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to 2, 1 to 3, 1 to 4, 2 to 50,5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6to 18), and each p is independently an integer from 1 to 10 (e.g., 1 to8, 1 to 6, 1 to 5, 1 to 3, 2 to 10, 4 to 10, 6 to 10, 2 to 8, and 3 to6).

In some embodiments, G is a hydrophobic separation handle. For example,G can be a substituted or unsubstituted trityloxy group. In someembodiments, G is selected from the group consisting of monoalkoxysubstituted trityloxy group or dialkoxy substituted trityloxy group.

In some embodiments, one of Z¹ and Z² is NH and the other is O. Forexample,

Z¹ is O and Z² is NH; Z¹ is NH and Z² is O. In some embodiments, both Z¹and Z² are O.

In some embodiments, L is a linear or branched alkylene. For example, Lcan be selected from the group consisting of: methylene, ethylene,propylene, isopropylene, butylene, isobutylene, sec-butylene,tert-butylene, and hexylene. In some embodiments, L can be analkyleneoxyalkylene or an oligomeric alkyleneoxyalkylene. For example, Lcan be a residue from diethylene glycol, triethylene glycol,tetraethylene glycol or hexaethylene glycol. In some embodiments, L isselected from the group consisting of —(CH₂)_(n)— and—((CH₂)_(n)—O—(CH₂)_(m))_(p)—, wherein n is an integer from 1 to 50(e.g., 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1to 10, 1 to 6, 1 to 6, 1 to 2, 1 to 3, 1 to 4, 2 to 50, 5 to 50, 10 to50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), m isan integer from 0 to 50 (e.g., 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to 2, 1 to 3, 1 to 4, 2 to 50,5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6to 18), and each p is independently an integer from 1 to 10 (e.g., 1 to8, 1 to 6, 1 to 5, 1 to 3, 2 to 10, 4 to 10, 6 to 10, 2 to 8, and 3 to6).

In some embodiments, L is a substituted or unsubstituted arylene. Forexample, L can be a structure with the formula:

where W is a substituent and r is an integer from 0 to 4. For example, Wcan be selected from the group consisting of: halo, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₅-C₁₂ aralkyl, C₃-C₁₂cycloalkyl, C₄-C₁₂ cycloalkenyl, phenyl, substituted phenyl, toluoyl,xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₅-C₁₂ alkoxyaryl, C₅-C₁₂aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀ alkylsulfonyl,—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8, aryl, substitutedaryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substitutedheterocyclic radical, nitroalkyl, —NO₂, —CN, —NR⁹C(O)—(C₁-C₁₀ alkyl),—C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkthioalkyl, —C(O)O—(C₁-C₁₀ alkyl), —OH,—SO₂, ═S, —COOH, —NR⁹ ₂, carbonyl, —C(O)—(C₁-C₁₀ alkyl)-CF₃, —C(O)—CF₃,—C(O)NR⁹ ₂, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀ aryl), —C(O)—(C₆-C₁₀ aryl),—(CH₂)_(m)—O—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein each m is from 1 to 8,—C(O)NR⁹ ₂, —C(S)NR⁹ ₂, —SO₂NR⁹ ₂, —NR⁹C(O)NR⁹ ₂, —NR⁹C(S)NR⁹ ₂, saltsthereof, and the like. Each R⁹ group in the preceding list independentlyincludes, but is not limited to, H, alkyl or substituted alkyl, aryl orsubstituted aryl, or alkylaryl. In some embodiments, W is R¹ asdescribed above.

Non-limiting examples of L include:

In some cases, L can also be a divalent radical of a nucleoside. Forexample, L can be a divalent radical of a natural nucleoside, such asadenosine, deoxyadenosine, guanosine, deoxyguanosine, 5-methyluridine,thymidine, uridine, deoxyuridine, cytidine, and deoxycytidine.

M is a group reactive with a biologically active molecule or aderivative thereof and can be selected from the group consisting of:hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl,alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide,aminoxy, maleimide, dithiopyridine, and iodoacetamide. In someembodiments, the group is protected or further reacted with a group R asshown in the structure of formula (1). The point of attachment of such agroup is well understood by those of skill in the art.

In some embodiments, R is absent. In some embodiments, R is a protectinggroup. For this purpose, R may include any suitable protecting groupbased on the group to be protected. For example, R may include anysuitable hydroxyl functional group including, but not limited to, ether,ester, carbonate, or sulfonate protecting groups.

In particular, the ether protecting group may include benzyloxymethyl(BOM), methylthiomethyl (MTM), phenylthiomethyl (PTM), cyanoethyl,2,2-dichloro-1,1-difluoroethyl, 2-chloroethyl, 2-bromoethyl,tetrahydropyranyl (THP), phenacyl, 4-bromophenacyl, allyl, propargyl,t-butyl, benzyl, 2,6-dimethylbenzyl, 4-methoxybenzyl (MPM-OAr),o-nitrobenzyl, 2,6-dichlorobenzyl, 3,4-dichlorobenzyl,4-(dimethylamino)carbonylbenzyl, 4-methylsulfinylbenzyl (Msib),9-anthrylemethyl, 4-picolyl, heptafluoro-p-tolyl, tetrafluoro-4-pyridyl,trimethylsilyl (TMS), and protecting groups.

The ester protecting group may include acetoxy (OAc), aryl formate,acetate, levulinate, pivaloate, benzoate, and 9-fluoroenecarboxylate. Inone embodiment, the ester protecting group is an acetoxy group.

The carbonate protecting group may include aryl methyl carbonate,1-adamantyl carbonate (Adoc-OAr), t-butyl carbonate (BOC—OAr),4-methylsulfinylbenzyl carbonate (Msz-OAr), 2,4-dimethylpent-3-ylcarbonate (Doc-OAr), aryl 2,2,2-trichloroethyl carbonate.

The sulfonate protecting groups may include aryl methanesulfonate, aryltoluenesulfonate, and aryl 2-formylbenzenesulfonate.

In some embodiments, R may include any suitable amino protecting group,including, but not limited to, carbamate, amide, N-alkyl, orN-aryl-derived protecting groups.

In particular, the carbamate protecting group may include, for example,9-fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc),carboxybenzyl carbamate (cbz), methyl carbamate, ethyl carbamate,9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethylcarbamate, 17-tetrabenzol[a,c,g,i]fluorenylmethyl carbamate (Tbfmoc),2-chloro-3-indenylmethyl carbamate (Climoc),2,7-di-t-butyl[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 1,1-dioxobenzo[b]thiophene-2-ylmethyl carbamate(Bsmoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethylcarbamate (Teoc), 2-phenylethyl carbamate (hZ), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-boc),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBoc),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc),N-2-pivaloylamino)-1,1-dimethylethyl carbamate,2-[(2-nitrophenyl)dithio]-1-phenylethyl carbamate (NpSSPeoc),2-(N,N-dicycloheylcarboxamido)ethyl carbamate, 1-adamanyl carbamate(1-Adoc), cinyl carbamate (Voc), 1-isopropylallyl carbamate (Ipaoc),4-nicrocinnamyl carbamate (Noc), 3-(3′pyridyl)prop-2-enyl carbamate(Paloc), 8-quinolyl carbamate, alkyldithio carbamate, p-methoxybenzylcarbamate (Moz), p-nitrobenzyl carbamate (Pnz), p-bromobenzyl carbamate,p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate,4-methylsulfinylbenzyl carbamate (Msz), diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 1,1-dimethyl-2-cyanoethyl carbamate, 2-(4-nitrophenyl)ethylcarbamate, 4-phenylacetoxybenzyl carbamate (PhAcOZ), andm-chloro-p-acyloxybenzyl carbamate. In some embodiments, the carbamateprotecting group is chosen from 9-fluorenylmethyl carbamate (Fmoc),t-butyl carbamate (Boc), and carboxybenzyl carbamate (cbz).

The amide protecting group may include, for example, acetamide,phenylacetamide, 3-phenylpropanamide, pent-4-enamide, picolinamide,3-pyridylcarboxamide, benzamide, p-phenylbenzamide,2-methyl-2-(o-phenylazophenoxy)propanamide), 4-chlorobutanamide,acetoacetamide, 3-(p-hydroxyphenyl)propanamide), and(N′-dithiobenzyloxycarbonylamino)acetamide.

Examples of suitable protecting groups also include tert-butyl, benzyl,4-methoxybenzyl, benzyloxymethyl, phenacyl, allyl, trimethylsilyl,benzyloxycarbonyl, tert-butoxycarbonyl, and acetal and ketalderivatives. In some embodiments, R is selected from trityls,substituted trityls (e.g., monomethoxytrityl (MMTr), dimethoxytrityl(DMTr), trimethoxytrityl (TMTr), 2-chlorotrityl (ClTr) andp-bromophenacyloxytrityl (BPTr), pixyls and substituted pixyls (see, forexample, U.S. Publication No. 2007/0276139). In some embodiments, R isselected from trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, andcyclic acetal.

In some embodiments, R¹ is a hydrophobic separation handle.

A hydrophobic separation handle is as described herein. In someembodiments, the hydrophobic separation handle is also a protectinggroup as described herein. In some embodiments, at least one of R, R¹,and G is a hydrophobic separation handle. For example, only one of R,R¹, and G is a hydrophobic separation handle.

In some embodiments, only one of R¹, R and G is a hydrophobic separationhandle (e.g., a trityl group) as provided herein. For example, if R is ahydrophobic separation handle, then R¹ is absent and G is hydrogen or analkoxy. In some embodiments, R is absent or a protecting group, R¹ is ahydrophobic separation handle, and G is hydrogen or an alkoxy. In someembodiments, wherein more than one of R, R¹ and G is a hydrophobicseparation handle, one of R, R¹ and G is more hydrophobic than theothers (e.g., substantially more hydrophobic). In some embodiments, thehydrophobic separation handle is a substituted or unsubstituted tritylor trityloxy group. For example, only one of R, R¹ and G is asubstituted or unsubstituted trityl or trityloxy group.

A compound as described above can be prepared, for example, bycontacting a water-soluble, non-peptidic and non-nucleotidic polymer, ina water-free solvent (e.g., an organic solvent), with a reagent offormula (4):

wherein:

R⁵ and R⁶ independently from each other represent C₁-C₆-alkyl or R⁵ andR⁶ jointly form a 5- or 6-membered ring with the N to which they arebonded. In some embodiments, R⁵ and R⁶ are independently a C₁-C₆-alkyl.For example, R⁵ and R⁶ can be independently selected from the groupconsisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, and hexyl. In some embodiments, R⁵ and R⁶ areisopropyl. In some embodiments, R⁵ and R⁶ can jointly form a 5- or6-membered ring with the N to which they are bonded. For example, R⁵ andR⁶ jointly form a pyrrolidine, pyrroline, imidazoline, pyrazolidine,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyridyl, pyrazinyl, pyrimidinyl,particularly 2- and 4-pyrimidinyl, pyridazinyl, pyrrolyl, particularly2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, or pyrazolyl, particularly3- and 5-pyrazolyl. In some embodiments, R⁵ and R⁶ can jointly form amorpholine ring.

The ratio of a polymer to a reagent of formula (4) can range from about1:10 to about 10:1 (e.g., about 2:1, about 3:1, about 4:1, about 5:1,about 6:1, about 7:1, about 8:1, about 9:1, about 1:2, about 1:3, about1:4, about 1:5, about 1:6, about 1:7, about 1:81 about 1:9, about 2:8,about 3:7, about 4:6 about 5:10, and about 4:8). In some embodiments,the ratio of a polymer to a reagent of formula (4) is from about 1:1 toabout 1:10, for example, about 2:1.

An activating reagent can then be added to the mixture of the polymerand the reagent of formula (4). An activating reagent can be any groupsuitable to initiate coupling of the polymer and the reagent of formula(4). Suitable activating reagents include, for example, 1H-tetrazole,5-(ethylthio)-1H-tetrazole (ETT), 5-(benzylthio)-1H-tetrazole (BTT),Activator 42 (5-(3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole),2-ethylthiotetrazole, 2-bezylthiotetrazole, 4,5-dicyanoimidazole and4,5-dicyanoimidazole (DCI). In some embodiments, an activating agent canbe selected from pyridinium hydrochloride, pyridinium trifluoroacetage,and buffered carboxylic acids.

An oxidizing agent can then be added to oxidize P⁺³ to P⁺⁵ Suitableoxidizing agents and conditions can be readily determined by those ofordinary skill in the art. For example, an oxidant such as RuO₄ ⁻/NMO,Dess-Martin's reagent, DMSO/triflic anhydride, PDC, hydrogen peroxide,inorganic peroxides, nitric acid, nitrates, chlorite, chlorate,perchlorate, hypochlorite, peroxides, iodine, ozone, nitrous oxide,silver oxide, permanganate salts, hexavalent chromium compounds, chromicacid, dichromic acids, chromium trioxide, pyridinium chlorochromate,persulfuric acid, sulfoxides, sulfuric acid, Tollens' reagent,2,2′-dipyridiyldisulfide (DPS), and osmium tetroxide may be used.

In some embodiments, iodine can be used as an oxidizing agent. Forexample, a solution of iodine can be used and prepared by dissolvingiodine in a mixture of pyridine, tetrahydrofuran and water. Elementalsulfur can be used for phosphite oxidation combined with formation ofsulfurized product. In some embodiments, other more soluble and morereactive reagents, such as 3H-1,2-benzothiazol-3-one 1,1-dioxide(Beaucage reagent), phenylacetyl disulfide (PADS), or dimethylthiuram(DTD) can be used. Alternatively, peroxides exemplified by t-butylhydrogen peroxide or m-chlorobenzoyl peroxide may be used for P⁺³ to P⁺⁵oxidations.

In some embodiments, an oxidizing reagent is selected from a groupconsisting of: iodine, hydrogen peroxide, t-butyl hydrogen peroxide,acetone peroxide, sulfur, and thiuram disulfide.

In some embodiments, R is a protecting group or a hydrophobic separationhandle and the method can include purifying the compound usingchromatography (e.g., reverse phase chromatography). In someembodiments, the method also includes removing the protecting group.

For the methods provided above, the deprotection may involve, forexample, either sequential or one-pot deprotection of certain protectinggroups. Suitable reagents and conditions for the deprotection can bereadily determined by those of ordinary skill in the art. For example,deprotection may be achieved upon treatment of the protected compoundunder conditions so that hydroxyl protecting groups, such as acetate,isopropylidine, benzylidine, trityl, and/or pixyl protecting groups, areremoved from the protected compound. The acetate group can be cleavedunder mild conditions, for example, by diluted solution of ammonia or bysolution of potassium carbonate. The benzylidene and isopropylidenegroups can be cleaved by hydrogenation or using acidic hydrolysis asdescribed elsewhere by R. M. Hann et al., J. Am. Chem. Soc., 72, 561(1950). In yet another example, the deprotection can be performed sothat amino protecting groups, such as 9-fluorenylmethyl carbamate(Fmoc), t-butyl carbamate (Boc), and carboxybenzyl carbamate (cbz)protecting groups are cleaved from the protected compound.9-fluorenylmethyl carbamate (FMOC) can be removed under mild conditionswith an amine base (e.g., piperidine) to afford the free amine anddibenzofulvene, as described by E. Atherton et al., “TheFluorenylmethoxycarbonyl Amino Protecting Group,” in The Peptides, S.Udenfriend and J. Meienhofer, Academic Press, New York, 1987, p. 1.t-butyl carbamate (Boc) can be removed, as reported by G. L. Stahl etal., J. Org. Chem., 43, 2285 (1978), under acidic conditions (e.g., 3 MHCl in EtOAc). Hydrogenation can be used to cleave the carboxybenzylcarbamate (cbz) protecting group as described by J. Meienhofer et al.,Tetrahedron Lett., 29, 2983 (1988).

In some embodiments, deprotection may be performed under anaerobicconditions. The deprotection may also be performed at ambienttemperature or at temperatures of from about 20-60° C. (e.g., 25, 30,35, 40, 45, 50, or 55° C.).

In some cases, a compound as described above can be further purifiedusing precipitation and/or crystallization.

Compounds of Formula (2)

Also provided herein are compounds of formula (2):

or a salt form thereof,

wherein:

polymer is a linear, water-soluble, non-peptidic, and non-nucleotidicpolymer backbone, wherein each linking group is bonded at a differentterminus of said polymer;

E and E¹ are independently O or S;

K and K₁ are independently selected from the group consisting of:alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene;

G and G₁ are independently absent or are selected from the groupconsisting of: alkoxy and a hydrophobic separation handle;

each pair of Z¹ and Z² and Z³ and Z⁴ are independently selected from Oand NH, wherein only one of each pair of Z¹ and Z² and Z³ and Z⁴ can beNH;

L and L¹ are independently selected from the group consisting of: adivalent radical of a nucleoside, alkylene, alkyleneoxyalkylene,oligomeric alkyleneoxyalkylene, and unsubstituted and substitutedarylene;

M and M¹ are independently selected from a protected group that whendeprotected is reactive with a biologically active molecule or aderivative thereof, a group reactive with a biologically active moleculeor a derivative thereof, or is a detectable functional group, wherein Mand M¹ are different, and wherein at least one of M and M1 is aprotected group that when deprotected is reactive with a biologicallyactive molecule or a derivative thereof or a group reactive with abiologically active molecule or a derivative thereof; and

R and R¹ are independently absent, hydrogen, a protecting group, or anactivating group;

wherein when M is a protected group that when deprotected is reactivewith a biologically active molecule or a derivative thereof, then R is aprotecting group or a hydrophobic separation handle;

wherein when M is a group reactive with a biologically active moleculeor a derivative thereof, R is absent, hydrogen, or an activating group;

wherein when M is a detectable functional group, R is absent orhydrogen;

wherein when M¹ is a protected group that when deprotected is reactivewith a biologically active molecule or a derivative thereof, then R¹ isa protecting group or a hydrophobic separation handle;

wherein when M¹ is a group reactive with a biologically active moleculeor a derivative thereof, R¹ is absent, hydrogen, or an activating group;and

wherein when M¹ is a detectable functional group, R¹ is absent orhydrogen.

A polymer can be, for example, poly(alkylene glycol), poly(oxyethylatedpolyol), poly(olefinic alcohol), poly(α-hydroxy acid), poly(vinylalcohol), polyoxazoline, or a copolymer thereof. Such polyalkyleneglycols, include, but are not limited to, polyethylene glycol,polypropylene glycol, polybutylene glycol, and derivatives thereof.Other exemplary embodiments are listed, for example, in commercialsupplier catalogs, such as Shearwater Corporation's catalog“Polyethylene Glycol and Derivatives for Biomedical Applications”(2001). By way of example only, such polymeric polyether polyols haveaverage molecular weights between about 0.1 kDa to about 100 kDa. By wayof example, such polymeric polyether polyols include, but are notlimited to, between about 500 Da and about 100,000 Da or more. Themolecular weight of the polymer may be between about 500 Da and about100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da,700 Da, 600 Da, and 500 Da. In some embodiments, the molecular weight ofthe polymer is between about 500 Da and about 50,000 Da. In someembodiments, the molecular weight of the polymer is between about 500 Daand about 40,000 Da. In some embodiments, the molecular weight of thepolymer is between about 1,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 5,000Da and about 40,000 Da. In some embodiments, the molecular weight of thepolymer is between about 10,000 Da and about 40,000 Da.

In some embodiments, the polymer is a poly(ethylene glycol) polymer. Themolecular weight of the PEG may be between about 1,000 Da and about100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In someembodiments, the molecular weight of the PEG is between about 1,000 Daand about 50,000 Da. In some embodiments, the molecular weight of the nPEG is between about 1,000 Da and about 40,000 Da. In some embodiments,the molecular weight of the PEG is between about 5,000 Da and about40,000 Da. In some embodiments, the molecular weight of the PEG isbetween about 5,000 Da and about 20,000 Da.

In some embodiments, E¹ is oxygen. In some embodiments, E¹ is sulfur. Insome embodiments, E² is oxygen. In some embodiments, E² is sulfur. Insome embodiments, both of E¹ and E² are oxygen.

In some embodiments, K and K¹ are independently selected from a linearor branched alkylene. For example, K and K¹ can be independentlyselected from the group consisting of: methylene, ethylene, propylene,isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, andhexylene. In some embodiments, K and K¹ are independently analkyleneoxyalkylene or an oligomeric alkyleneoxyalkylene. For example, Kand K¹ can be independently a residue from diethylene glycol,triethylene glycol, tetraethylene glycol or hexaethylene glycol. In someembodiments, K and K¹ are independently selected from the groupconsisting of —(CH₂)_(n)— and —((CH₂)_(n)—O—(CH₂)_(m))_(p)—, wherein nis an integer from 1 to 50 (e.g., 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1to 18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to 6, 1 to 2, 1 to 3, 1 to4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20to 30, and 6 to 18), m is an integer from 0 to 50 (e.g., 1 to 40, 1 to30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to2, 1 to 3, 1 to 4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to15, 2 to 12, 20 to 30, and 6 to 18), and each p is independently aninteger from 1 to 10 (e.g., 1 to 8, 1 to 6, 1 to 5, 1 to 3, 2 to 10, 4to 10, 6 to 10, 2 to 8, and 3 to 6).

In some embodiments, G and G¹ are independently a hydrophobic separationhandle. For example, G and G¹ are independently a substituted orunsubstituted trityloxy group. In some embodiments, G and G¹ areindependently selected from the group consisting of monoalkoxysubstituted trityloxy group or dialkoxy substituted trityloxy group.

In some embodiments, one of Z¹ and Z² is NH and the other is O. Forexample, Z¹ is O and Z² is NH; Z¹ is NH and Z² is O. In someembodiments, both Z¹ and Z² are O. In some embodiments, one of Z¹ and Z²is NH and the other is O. For example, Z³ is O and Z⁴ is NH; Z³ is NHand Z⁴ is O. In some embodiments, both Z³ and Z⁴ are O. In someembodiments, one of Z¹ and Z² and Z³ and Z⁴ is NH and the other is O.For example, Z¹ and Z³ are O and Z² and Z⁴ are NH; Z¹ and Z³ are NH andZ² and Z⁴O. In some embodiments, Z¹ and Z³ are O and Z² and Z⁴ are O.

In some embodiments, L and L¹ are independently selected from a linearor branched alkyl. For example, L and L¹ can be independently selectedfrom the group consisting of: methylene, ethylene, propylene,isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, andhexylene. In some embodiments, L and L¹ are independently analkyleneoxyalkylene or an oligomeric alkyleneoxyalkylene. For example, Land L¹ can be independently a residue from diethylene glycol,triethylene glycol, tetraethylene glycol or hexaethylene glycol. In someembodiments, L and L¹ are independently selected from the groupconsisting of —(CH₂)_(n)— and —((CH₂)_(n)—O—(CH₂)_(m))_(p)—, wherein nis an integer from 1 to 50 (e.g., 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1to 18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to 6, 1 to 2, 1 to 3, 1 to4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20to 30, and 6 to 18), m is an integer from 0 to 50 (e.g., 1 to 40, 1 to30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to2, 1 to 3, 1 to 4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to15, 2 to 12, 20 to 30, and 6 to 18), and each p is independently aninteger from 1 to 10 (e.g., 1 to 8, 1 to 6, 1 to 5, 1 to 3, 2 to 10, 4to 10, 6 to 10, 2 to 8, and 3 to 6).

In some embodiments, L and L¹ are independently a substituted orunsubstituted arylene. For example, L and L¹ can be independently astructure with the formula:

wherein W is a substituent and r is an integer from 0 to 4. For example,W can be selected from the group consisting of: halo, C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₅-C₁₂ aralkyl, C₃-C₁₂cycloalkyl, C₄-C₁₂ cycloalkenyl, phenyl, substituted phenyl, toluoyl,xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₅-C₁₂ alkoxyaryl, C₅-C₁₂aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀ alkylsulfonyl,—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8, aryl, substitutedaryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substitutedheterocyclic radical, nitroalkyl, —NO₂, —CN, —NR⁹C(O)—(C₁-C₁₀ alkyl),C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkthioalkyl, —C(O)O—(C₁-C₁₀ alkyl), —OH,—SO₂, ═S, COOH, —NR⁹ ₂, carbonyl, —C(O)—(C₁-C₁₀ alkyl)-CF₃, —C(O)—CF₃,—C(O)NR⁹ ₂, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀ aryl), —C(O)—(C₆-C₁₀ aryl),—(CH₂)_(m)—O—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein each m is from 1 to 8,—C(O)NR⁹ ₂, —C(S)NR⁹ ₂, —SO₂NR⁹ ₂, —NR⁹C(O)NR⁹ ₂, NR⁹C(S)NR⁹ ₂, saltsthereof, and the like. Each R⁹ group in the preceding list independentlyincludes, but is not limited to, H, alkyl or substituted alkyl, aryl orsubstituted aryl, or alkylaryl. In some embodiments, W is R¹ asdescribed above.

Non-limiting examples of L and L¹ include:

L and L¹ can also independently be a divalent radical of a nucleoside.For example, L and L¹ can be a divalent radical of a natural nucleoside,such as adenosine, deoxyadenosine, guanosine, deoxyguanosine,5-methyluridine, thymidine, uridine, deoxyuridine, cytidine, anddeoxycytidine.

M is a group reactive with a biologically active molecule or aderivative thereof and can be selected from the group consisting of:hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl,alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide,aminoxy, maleimide, dithiopyridine, and iodoacetamide. In someembodiments, the group is protected or further reacted with a group Rand R¹ as shown in the formula (2). The point of attachment of such agroup is well understood by those of skill in the art.

In some embodiments, M or M¹ is a detectable functional group. Adetectable functional group, as used herein, can be any chemical orsubstance which is used to provide a signal or contrast in imaging. Thesignal enhancing domain can be an organic molecule, metal ion, salt orchelate, particle (particularly iron particle), or labeled peptide,protein, polymer or liposome. For example, a detectable functional groupcan include one or more of a radionuclide, a paramagnetic metal, afluorophore, a dye, and an enzyme substrate. In some embodiments, adetectable functional group is biotin or a fluorophore.

In some embodiments, the detectable functional group is aphysiologically compatible metal chelate compound consisting of one ormore cyclic or acyclic organic chelating agents complexed to one or moremetal ions with atomic numbers 21-29, 42, 44, or 57-83.

For x-ray imaging, the detectable functional group may consist ofiodinated organic molecules or chelates of heavy metal ions of atomicnumbers 57 to 83. In some embodiments, the detectable functional groupis ¹²⁵I-IgG. Examples of suitable compounds are described in M. Sovak,ed., “Radiocontrast Agents,” Springer-Verlag, pp. 23-125 (1984) and U.S.Pat. No. 4,647,447.

For ultrasound imaging, the detectable functional group can consist ofgas-filled bubbles such as Albunex, Echovist, or Levovist, or particlesor metal chelates where the metal ions have atomic numbers 21-29, 42, 44or 57-83. Examples of suitable compounds are described in Tyler et al.,Ultrasonic Imaging, 3, pp. 323-29 (1981) and D. P. Swanson, “EnhancementAgents for Ultrasound: Fundamentals,” Pharmaceuticals in MedicalImaging, pp. 682-87. (1990).

For nuclear radiopharmaceutical imaging or radiotherapy, the detectablefunctional group can consist of a radioactive molecule. In someembodiments, the chelates of Tc, Re, Co, Cu, Au, Ag, Pb, Bi, In, and Gacan be used. In some embodiments, the chelates of Tc-99m can be used.Examples of suitable compounds are described in Rayudu G V S,Radiotracers for Medical Applications, I, pp. 201 and D. P. Swanson etal., ed., Pharmaceuticals in Medical Imaging, pp. 279-644 (1990).

For ultraviolet/visible/infrared light imaging, the detectablefunctional group can consist of any organic or inorganic dye or anymetal chelate.

For MRI, the detectable functional group can consist of a metal-ligandcomplex of a paramagnetic form of a metal ion with atomic numbers 21-29,42, 44, or 57-83. In some embodiments, the paramagnetic metal is chosenfrom: Gd(III), Fe(III), Mn(II and III), Cr(III), Cu(II), Dy(III),Tb(III), Ho(III), Er(III) and Eu(III). Many suitable chelating ligandsfor MRI agents are known in the art. These can also be used for metalchelates for other forms of biological imaging. For example, an imagingagent can include: Gadovist, Magnevist, Dotarem, Omniscan, and ProHance.

In some embodiments, R and/or R¹ is absent. In some embodiments, Rand/or R¹ is a protecting group. For this purpose, R and/or R¹ mayinclude any suitable protecting group based on the group to beprotected. For example, R and/or R¹ may include any suitable hydroxylfunctional group including, but not limited to, ether, ester, carbonate,or sulfonate protecting groups.

In particular, the ether protecting group may include benzyloxymethyl(BOM), methylthiomethyl (MTM), phenylthiomethyl (PTM), cyanoethyl,2,2-dichloro-1,1-difluoroethyl, 2-chloroethyl, 2-bromoethyl,tetrahydropyranyl (THP), phenacyl, 4-bromophenacyl, allyl, propargyl,t-butyl, benzyl, 2,6-dimethylbenzyl, 4-methoxybenzyl (MPM-OAr),o-nitrobenzyl, 2,6-dichlorobenzyl, 3,4-dichlorobenzyl,4-(dimethylamino)carbonylbenzyl, 4-methylsulfinylbenzyl (Msib),9-anthrylemethyl, 4-picolyl, heptafluoro-p-tolyl, tetrafluoro-4-pyridyl,trimethylsilyl (TMS), and protecting groups.

The ester protecting group may include acetoxy (OAc), aryl formate,acetate, levulinate, pivaloate, benzoate, and 9-fluoroenecarboxylate. Inone embodiment, the ester protecting group is an acetoxy group.

The carbonate protecting group may include aryl methyl carbonate,1-adamantyl carbonate (Adoc-OAr), t-butyl carbonate (BOC—OAr),4-methylsulfinylbenzyl carbonate (Msz-OAr), 2,4-dimethylpent-3-ylcarbonate (Doc-OAr), aryl 2,2,2-trichloroethyl carbonate.

The sulfonate protecting groups may include aryl methanesulfonate, aryltoluenesulfonate, and aryl 2-formylbenzenesulfonate.

In some embodiments, R may include any suitable amino protecting group,including, but not limited to, carbamate, amide, N-alkyl, orN-aryl-derived protecting groups.

In particular, the carbamate protecting group may include, for example,9-fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc),carboxybenzyl carbamate (cbz), methyl carbamate, ethyl carbamate,9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethylcarbamate, 17-tetrabenzol[a,c,g,i]fluorenylmethyl carbamate (Tbfmoc),2-chloro-3-indenylmethyl carbamate (Climoc),2,7-di-t-butyl[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 1,1-dioxobenzo[b]thiophene-2-ylmethyl carbamate(Bsmoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethylcarbamate (Teoc), 2-phenylethyl carbamate (hZ), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-boc),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBoc),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc),N-2-pivaloylamino)-1,1-dimethylethyl carbamate,2-[(2-nitrophenyl)dithio]-1-phenylethyl carbamate (NpSSPeoc),2-(N,N-dicycloheylcarboxamido)ethyl carbamate, 1-adamanyl carbamate(1-Adoc), cinyl carbamate (Voc), 1-isopropylallyl carbamate (Ipaoc),4-nicrocinnamyl carbamate (Noc), 3-(3′pyridyl)prop-2-enyl carbamate(Paloc), 8-quinolyl carbamate, alkyldithio carbamate, p-methoxybenzylcarbamate (Moz), p-nitrobenzyl carbamate (Pnz), p-bromobenzyl carbamate,p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate,4-methylsulfinylbenzyl carbamate (Msz), diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 1,1-dimethyl-2-cyanoethyl carbamate, 2-(4-nitrophenyl)ethylcarbamate, 4-phenylacetoxybenzyl carbamate (PhAcOZ), andm-chloro-p-acyloxybenzyl carbamate. In some embodiments, the carbamateprotecting group is chosen from 9-fluorenylmethyl carbamate (Fmoc),t-butyl carbamate (Boc), and carboxybenzyl carbamate (cbz).

The amide protecting group may include, for example, acetamide,phenylacetamide, 3-phenylpropanamide, pent-4-enamide, picolinamide,3-pyridylcarboxamide, benzamide, p-phenylbenzamide,2-methyl-2-(o-phenylazophenoxy)propanamide), 4-chlorobutanamide,acetoacetamide, 3-(p-hydroxyphenyl)propanamide), and(N′-dithiobenzyloxycarbonylamino)acetamide.

Examples of suitable protecting groups also include tert-butyl, benzyl,4-methoxybenzyl, benzyloxymethyl, phenacyl, allyl, trimethylsilyl,benzyloxycarbonyl, tert-butoxycarbonyl, and acetal and ketalderivatives. In some embodiments, R is selected from trityls,substituted trityls (e.g., monomethoxytrityl (MMTr), dimethoxytrityl(DMTr), trimethoxytrityl (TMTr), 2-chlorotrityl (ClTr) andp-bromophenacyloxytrityl (BPTr), pixyls and substituted pixyls (see, forexample, U.S. Publication No. 2007/0276139). In some embodiments, R isselected from trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, andcyclic acetal.

In some embodiments, R and/or R¹ is a hydrophobic separation handle.

A hydrophobic separation handle is as described herein. In someembodiments, the hydrophobic separation handle is also a protectinggroup as described herein. In some embodiments, at least one of R, R¹,G, and G¹ is a hydrophobic separation handle.

In some embodiments, only one of R and G and R¹ and G¹ is a hydrophobicseparation handle (e.g., a trityl group) as provided herein. Forexample, if R is a hydrophobic separation handle, then G is hydrogen oran alkoxy. Alternatively, if R¹ is a hydrophobic separation handle, thenG¹ is absent or an alkoxy. In some embodiments, R is absent or aprotecting group, R¹ is a hydrophobic separation handle, and G and G¹are independently absent or an alkoxy, wherein R¹ is more hydrophobicthan R. In some embodiments, R¹ is absent or a protecting group, R is ahydrophobic separation handle, and G and G¹ are independently absent oran alkoxy, wherein R is more hydrophobic than R¹. In some embodiments,the hydrophobic separation handle is a substituted or unsubstitutedtrityl or trityloxy group. For example, only one of R, R¹, G, and G¹ isa substituted or unsubstituted trityl or trityloxy group.

A compound as described above can be prepared, for example, bycontacting a water-soluble, non-peptidic and non-nucleotidic polymer, ina water-free solvent (e.g., an organic solvent), with a reagent selectedfrom formula (5):

wherein:

R⁵ and R⁶ independently from each other represent C₁-C₆-alkyl or R⁵ andR⁶ jointly form a 5- or 6-membered ring with the N to which they arebonded, and formula (6):

wherein:

R⁷ and R⁸ independently from each other represent C₁-C₆-alkyl or R⁷ andR⁸ jointly form a 5- or 6-membered ring with the N to which they arebonded;

under conditions that facilitate formation of monoderivatized product.

In some embodiments, R⁵ and R⁶ are independently a C₁-C₆-alkyl. Forexample, R⁵ and R⁶ can be independently selected from the groupconsisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, and hexyl. In some embodiments, R⁵ and R⁶ areisopropyl. In some embodiments, R⁵ and R⁶ jointly form a 5- or6-membered ring with the N to which they are bonded. For example, R⁵ andR⁶ jointly form a pyrrolidine, pyrroline, imidazoline, pyrazolidine,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyridyl, pyrazinyl, pyrimidinyl,particularly 2- and 4-pyrimidinyl, pyridazinyl, pyrrolyl, particularly2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, and pyrazolyl, particularly3- and 5-pyrazolyl. In some embodiments, R⁵ and R⁶ jointly form amorpholine ring.

In some embodiments, R⁷ and R⁸ are independently a C₁-C₆-alkyl. Forexample, R⁷ and R⁸ can be independently selected from the groupconsisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, and hexyl. In some embodiments, R⁷ and R⁸ areisopropyl. In some embodiments, R⁷ and R⁸ jointly form a 5- or6-membered ring with the N to which they are bonded. For example, R⁷ andR⁸ jointly form a pyrrolidine, pyrroline, imidazoline, pyrazolidine,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyridyl, pyrazinyl, pyrimidinyl,particularly 2- and 4-pyrimidinyl, pyridazinyl, pyrrolyl, particularly2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, and pyrazolyl, particularly3- and 5-pyrazolyl. In some embodiments, R⁷ and R⁸ jointly form amorpholine ring.

The ratio of a polymer to a reagent of formula (5) or (6) can range fromabout 1:10 to about 10:1 (e.g., about 2:1, about 3:1, about 4:1, about5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 1:2, about 1:3,about 1:4, about 1:5, about 1:6, about 1:7, about 1:81 about 1:9, about2:8, about 3:7, about 4:6 about 5:10, and about 4:8). In someembodiments, the ratio of a polymer to a reagent of formula (5) or (6)is from about 1:1 to about 1:10. In some embodiments, the ratio of apolymer to a reagent of formula (5) or (6) is about 2:1.

In some embodiments, conditions that facilitate formation of amonoderivatized product include the addition of an activating reagent.An activating reagent is then added to the mixture of the polymer andthe reagent of formula (4) or (5). An activating reagent can be anygroup suitable to initiate coupling of the polymer and the reagent offormula (4). Suitable activating reagents include, for example,1H-tetrazole, 5-(ethylthio)-1H-tetrazole (ETT),5-(benzylthio)-1H-tetrazole (BTT), Activator 42(5-(3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole), 2-ethylthiotetrazole,2-bezylthiotetrazole, 4,5-dicyanoimidazole and 4,5-dicyanoimidazole(DCI). In some embodiments, an activating agent can be selected frompyridinium hydrochloride, pyridinium trifluoroacetage, and bufferedcarboxylic acids.

In some embodiments, conditions that facilitate formation of amonoderivatized product include addition of an oxidizing agent tooxidize P⁺³ to P⁺⁵. Suitable oxidizing agents and conditions can bereadily determined by those of ordinary skill in the art. For example,an oxidant such as RuO₄ ⁻/NMO, Dess-Martin's reagent, DMSO/triflicanhydride, PDC, hydrogen peroxide, inorganic peroxides, nitric acid,nitrates, chlorite, chlorate, perchlorate, hypochlorite, peroxide,iodine, ozone, nitrous oxide, silver oxide, permanganate salts,hexavalent chromium compounds, chromic acid, dichromic acids, chromiumtrioxide, pyridinium chlorochromate, persulfuric acid, sulfoxides,sulfuric acid, Tollens' reagent, 2,2′-dipyridiyldisulfide (DPS), andosmium tetroxide may be used.

In some embodiments, iodine can be used as an oxidizing agent. Forexample, a solution of iodine can be used and prepared by dissolvingiodine in a mixture of pyridine, tetrahydrofuran and water. Elementalsulfur can be used for phosphite oxidation combined with formation ofsulfurized product. In some embodiments, other more soluble and morereactive reagents, such as 3H-1,2-benzothiazol-3-one 1,1-dioxide(Beaucage reagent), phenylacetyl disulfide (PADS) or dimethylthiuram(DTD) can be used. Alternatively, peroxides exemplified by t-butylhydrogen peroxide or m-chlorobenzoyl peroxide may be used for P⁺³ to P⁺⁵oxidations.

In some embodiments, an oxidizing reagent is selected from a groupconsisting of: iodine, hydrogen peroxide, t-butyl hydrogen peroxide,acetone peroxide, sulfur, and thiuram disulfide.

In some embodiments, R and/or R¹ is a protecting group or a hydrophobicseparation handle. In some embodiments, the method can further includepurifying the monoderivatized compound using chromatography (e.g.,reverse phase chromatography).

To the monoderivatized product, a reagent of formula (4) or formula (5)is added under conditions that facilitate the conversion of themonoderivatized product to a compound of formula (2). In someembodiments, the reagent is different from the reagent used to preparethe monoderivatized product. In some embodiments, the reagent is thesame as that used to prepare the monoderivatized product.

In some embodiments, the conditions are such that conversion of themonoderivatized product to the compound of formula (2) is quantitative.

In some embodiments, conditions that facilitate formation of a compoundof formula (2) include the addition of an activating reagent. Anactivating reagent is then added to the mixture of the monoderivatizedproduct and the reagent of formula (5) or (6). An activating reagent canbe any group suitable to initiate coupling of the polymer and thereagent of formula (5) or (6). Suitable activating reagents include, forexample, 1H-tetrazole, 5-(ethylthio)-1H-tetrazole (ETT),5-(benzylthio)-1H-tetrazole (BTT), Activator 42(5-(3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole), 2-ethylthiotetrazole,2-bezylthiotetrazole, 4,5-dicyanoimidazole and 4,5-dicyanoimidazole(DCI). In some embodiments, an activating agent can be selected frompyridinium hydrochloride, pyridinium trifluoroacetage, and bufferedcarboxylic acids.

In some embodiments, conditions that facilitate formation of a compoundof formula (2) include addition of an oxidizing agent to oxidize P⁺³ toP⁺⁵. Suitable oxidizing agents and conditions can be readily determinedby those of ordinary skill in the art. For example, an oxidant such asRuO₄ ⁻/NMO, Dess-Martin's reagent, DMSO/triflic anhydride, PDC, hydrogenperoxide, inorganic peroxides, nitric acid, nitrates, chlorite,chlorate, perchlorate, hypochlorite, peroxide, iodine, ozone, nitrousoxide, silver oxide, permanganate salts, hexavalent chromium compounds,chromic acid, dichromic acids, chromium trioxide, pyridiniumchlorochromate, persulfuric acid, sulfoxides, sulfuric acid, Tollens'reagent, 2,2′-dipyridiyldisulfide (DPS), and osmium tetroxide may beused.

In some embodiments, iodine can be used as an oxidizing agent. Forexample, a solution of iodine can be used and prepared by dissolvingiodine in a mixture of pyridine, tetrahydrofuran and water. Elementalsulfur can be used for phosphite oxidation combined with formation ofsulfurized product. In some embodiments, other more soluble and morereactive reagents, such as 3H-1,2-benzothiazol-3-one 1,1-dioxide(Beaucage reagent), phenylacetyl disulfide (PADS) or dimethylthiuram(DTD) can be used. Alternatively, peroxides exemplified by t-butylhydrogen peroxide or m-chlorobenzoyl peroxide may be used for P⁺³ to P⁺⁵oxidations.

In some embodiments, an oxidizing reagent is selected from a groupconsisting of: iodine, hydrogen peroxide, t-butyl hydrogen peroxide,acetone peroxide, sulfur, and thiuram disulfide.

In some embodiments, R and/or R¹ is a protecting group or a hydrophobicseparation handle. In some embodiments, the method can further includepurifying the monoderivatized compound using chromatography (e.g.,reverse phase chromatography). In some embodiments, the method furtherincludes removal of one or more of the protecting groups. In someembodiments, the method further includes removal of one or more of thehydrophobic separation handles.

For the methods provided above, the deprotection may involve, forexample, either sequential or one-pot deprotection of certain protectinggroups. Suitable reagents and conditions for the deprotection can bereadily determined by those of ordinary skill in the art. For example,deprotection may be achieved upon treatment of the protected compoundunder conditions so that hydroxyl protecting groups, such as acetate,isopropylidine, benzylidine, trityl, and pixyl protecting groups, areremoved from the protected compound. The acetate group can be cleavedunder mild conditions, for example, by diluted solution of ammonia or bysolution of potassium carbonate. The benzylidene and isopropylidenegroups can be cleaved by hydrogenation or using acidic hydrolysis asreported by R. M. Hann et al., J. Am. Chem. Soc., 72, 561 (1950). In yetanother example, the deprotection can be performed so that aminoprotecting groups, such as 9-fluorenylmethyl carbamate (Fmoc), t-butylcarbamate (Boc), and carboxybenzyl carbamate (cbz) protecting groups arecleaved from the protected compound. 9-fluorenylmethyl carbamate (FMOC)can be removed under mild conditions with an amine base (e.g.,piperidine) to afford the free amine and dibenzofulvene, as described byE. Atherton et al., “The Fluorenylmethoxycarbonyl Amino ProtectingGroup,” in The Peptides, S. Udenfriend and J. Meienhofer, AcademicPress, New York, 1987, p. 1. t-butyl carbamate (Boc) can be removed, asreported by G. L. Stahl et al., J. Org. Chem., 43, 2285 (1978), underacidic conditions (e.g., 3 M HCl in EtOAc). Hydrogenation can be used tocleave the carboxybenzyl carbamate (cbz) protecting group as describedby J. Meienhofer et al., Tetrahedron Lett., 29, 2983 (1988).

In some embodiments, deprotection may be performed under anaerobicconditions. The deprotection may also be performed at ambienttemperature or at temperatures of from about 20-60° C. (e.g., 25, 30,35, 40, 45, 50, or 55° C.).

In some embodiments, the method can also include isolating the compoundby precipitation or crystallization.

Compounds of Formula (3)

Also provided herein is a compound of formula (3):

or a salt form thereof,

wherein:

polymer is a linear, water-soluble, non-peptidic, and non-nucleotidicpolymer backbone, wherein M² and the phosphonate-derived functionalgroup are bonded at a different terminus of said polymer;

E and E¹ are independently O or S;

K is selected from the group consisting of: alkylene,alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene;

G is selected from the group consisting of hydrogen, alkoxy and ahydrophobic separation handle;

Z¹ and Z² are independently selected from O and NH, wherein only one ofZ¹ and Z² can be NH;

L is selected from the group consisting of: a divalent radical of anucleoside, alkylene, alkyleneoxyalkylene, oligomericalkyleneoxyalkylene, and unsubstituted and substituted arylene;

M is selected from a protected group that when deprotected is reactivewith a biologically active molecule or a derivative thereof or a groupreactive with a biologically active molecule;

M² is selected from O, S or NH; and

R is absent, a protecting group, a hydrophobic separation handle, or anactivating group;

R² is hydrogen or a protecting group;

wherein when M is a protected group that when deprotected is reactivewith a biologically active molecule or a derivative thereof, then R is aprotecting group or a hydrophobic separation handle; and

wherein when M is a group reactive with a biologically active moleculeor a derivative thereof, R is absent, hydrogen, or an activating group;and

A polymer can be, for example, poly(alkylene glycol), poly(oxyethylatedpolyol), poly(olefinic alcohol), poly (•-hydroxy acid), poly(vinylalcohol), polyoxazoline, or a copolymer thereof. Such polyalkyleneglycols, include, but are not limited to, polyethylene glycol,polypropylene glycol, polybutylene glycol, and derivatives thereof.Other exemplary embodiments are listed, for example, in commercialsupplier catalogs, such as Shearwater Corporation's catalog“Polyethylene Glycol and Derivatives for Biomedical Applications”(2001). By way of example only, such polymeric polyether polyols haveaverage molecular weights between about 0.1 kDa to about 100 kDa. By wayof example, such polymeric polyether polyols include, but are notlimited to, between about 500 Da and about 100,000 Da or more. Themolecular weight of the polymer may be between about 500 Da and about100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da,700 Da, 600 Da, and 500 Da. In some embodiments, the molecular weight ofthe polymer is between about 500 Da and about 50,000 Da. In someembodiments, the molecular weight of the polymer is between about 500 Daand about 40,000 Da. In some embodiments, the molecular weight of thepolymer is between about 1,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 5,000Da and about 40,000 Da. In some embodiments, the molecular weight of thepolymer is between about 10,000 Da and about 40,000 Da.

In some embodiments, the polymer is a poly(ethylene glycol) polymer. Themolecular weight of the PEG may be between about 1,000 Da and about100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In someembodiments, the molecular weight of the PEG is between about 1,000 Daand about 50,000 Da. In some embodiments, the molecular weight of the nPEG is between about 1,000 Da and about 40,000 Da. In some embodiments,the molecular weight of the PEG is between about 5,000 Da and about40,000 Da. In some embodiments, the molecular weight of the PEG isbetween about 5,000 Da and about 20,000 Da.

In some embodiments, E is oxygen. In some embodiments, E is sulfur.

In some embodiments, K is a linear or branched alkylene. For example, Kcan be selected from the group consisting of: methylene, ethylene,propylene, isopropylene, butylene, isobutylene, sec-butylene,tert-butylene, and hexylene. In some embodiments, K can be analkyleneoxyalkylene or an oligomeric alkyleneoxyalkylene. For example, Kcan be a residue from diethylene glycol, triethylene glycol,tetraethylene glycol or hexaethylene glycol. In some embodiments, K isselected from the group consisting of —(CH₂)_(n)— and—((CH₂)_(n)—O—(CH₂)_(m))_(p)—, wherein n is an integer from 1 to 50(e.g., 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1to 10, 1 to 6, 1 to 6, 1 to 2, 1 to 3, 1 to 4, 2 to 50, 5 to 50, 10 to50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), m isan integer from 0 to 50 (e.g., 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to 2, 1 to 3, 1 to 4, 2 to 50,5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6to 18), and each p is independently an integer from 1 to 10 (e.g., 1 to8, 1 to 6, 1 to 5, 1 to 3, 2 to 10, 4 to 10, 6 to 10, 2 to 8, and 3 to6).

In some embodiments, G is a hydrophobic separation handle. For example,G can be a substituted or unsubstituted trityloxy group. In someembodiments, G is selected from the group consisting of monoalkoxysubstituted trityloxy group or dialkoxy substituted trityloxy group.

In some embodiments, one of Z¹ and Z² is NH and the other is O. Forexample,

Z¹ is O and Z² is NH; Z¹ is NH and Z² is O. In some embodiments, both Z¹and Z² are O.

In some embodiments, L is a linear or branched alkyl. For example, L canbe selected from the group consisting of: methylene, ethylene,propylene, isopropylene, butylene, isobutylene, sec-butylene,tert-butylene, and hexylene. In some embodiments, L can be analkyleneoxyalkylene or an oligomeric alkyleneoxyalkylene. For example, Lcan be a residue from diethylene glycol, triethylene glycol,tetraethylene glycol or hexaethylene glycol. In some embodiments, L isselected from the group consisting of —(CH₂)_(n)— and—((CH₂)_(n)—O—(CH₂)_(m))_(p)—, wherein n is an integer from 1 to 50(e.g., 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1to 10, 1 to 6, 1 to 6, 1 to 2, 1 to 3, 1 to 4, 2 to 50, 5 to 50, 10 to50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), m isan integer from 0 to 50 (e.g., 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to 2, 1 to 3, 1 to 4, 2 to 50,5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6to 18), and each p is independently an integer from 1 to 10 (e.g., 1 to8, 1 to 6, 1 to 5, 1 to 3, 2 to 10, 4 to 10, 6 to 10, 2 to 8, and 3 to6).

In some embodiments, L is a substituted or unsubstituted arylene. Forexample, L can be a structure with the formula:

wherein W is a substituent and r is an integer from 0 to 4. For example,W can be selected from the group consisting of: halo, C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₅-C₁₂ aralkyl, C₃-C₁₂cycloalkyl, C₄-C₁₂ cycloalkenyl, phenyl, substituted phenyl, toluoyl,xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₅-C₁₂ alkoxyaryl, C₅-C₁₂aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀ alkylsulfonyl,—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8, aryl, substitutedaryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substitutedheterocyclic radical, nitroalkyl, —NO₂, —CN, —NR⁹C(O)—(C₁-C₁₀ alkyl),—C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkthioalkyl, —C(O)O—(C₁-C₁₀ alkyl), —OH,—SO₂, ═S, —COOH, —NR⁹ ₂, carbonyl, —C(O)—(C₁-C₁₀ alkyl)-CF₃, —C(O)—CF₃,—C(O)NR⁹ ₂, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀ aryl), —C(O)—(C₆-C₁₀ aryl),—(CH₂)_(m)—O—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein each m is from 1 to 8,—C(O)NR⁹ ₂, —C(S)NR⁹ ₂, —SO₂NR⁹ ₂, —NR⁹C(O)NR⁹ ₂, NR⁹C(S)NR⁹ ₂, saltsthereof, and the like. Each R⁹ group in the preceding list independentlyincludes, but is not limited to, H, alkyl or substituted alkyl, aryl orsubstituted aryl, or alkylaryl. In some embodiments, W is R¹ asdescribed above.

Non-limiting examples of L include:

L can also be a divalent radical of a nucleoside. For example, L can bea divalent radical of a natural nucleoside, such as adenosine,deoxyadenosine, guanosine, deoxyguanosine, 5-methyluridine, thymidine,uridine, deoxyuridine, cytidine, and deoxycytidine.

A group reactive with a biologically active molecule or a derivativethereof can be selected from the group consisting of: hydroxyl, amine,thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl,azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide,dithiopyridine, iodoacetamide. In some embodiments, the group isprotected or further reacted with a group R as shown in the structure offormula (3). The point of attachment of such a group is well understoodby those of skill in the art.

In some embodiments, R is absent.

In some embodiments, R² is hydrogen.

In some embodiments, R and/or R² is a protecting group. For thispurpose, R and/or R² may include any suitable protecting group based onthe group to be protected. For example, R and/or R² may include anysuitable hydroxyl functional group including, but not limited to, ether,ester, carbonate, or sulfonate protecting groups.

In particular, the ether protecting group may include benzyloxymethyl(BOM), methylthiomethyl (MTM), phenylthiomethyl (PTM), cyanoethyl,2,2-dichloro-1,1-difluoroethyl, 2-chloroethyl, 2-bromoethyl,tetrahydropyranyl (THP), phenacyl, 4-bromophenacyl, allyl, propargyl,t-butyl, benzyl, 2,6-dimethylbenzyl, 4-methoxybenzyl (MPM-OAr),o-nitrobenzyl, 2,6-dichlorobenzyl, 3,4-dichlorobenzyl,4-(dimethylamino)carbonylbenzyl, 4-methylsulfinylbenzyl (Msib),9-anthrylemethyl, 4-picolyl, heptafluoro-p-tolyl, tetrafluoro-4-pyridyl,trimethylsilyl (TMS), and protecting groups.

The ester protecting group may include acetoxy (OAc), aryl formate,acetate, levulinate, pivaloate, benzoate, and 9-fluoroenecarboxylate. Inone embodiment, the ester protecting group is an acetoxy group.

The carbonate protecting group may include aryl methyl carbonate,1-adamantyl carbonate (Adoc-OAr), t-butyl carbonate (BOC—OAr),4-methylsulfinylbenzyl carbonate (Msz-OAr), 2,4-dimethylpent-3-ylcarbonate (Doc-OAr), aryl 2,2,2-trichloroethyl carbonate.

The sulfonate protecting groups may include aryl methanesulfonate, aryltoluenesulfonate, and aryl 2-formylbenzenesulfonate.

In some embodiments, R may include any suitable amino protecting group,including, but not limited to, carbamate, amide, N-alkyl, orN-aryl-derived protecting groups.

In particular, the carbamate protecting group may include, for example,9-fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc),carboxybenzyl carbamate (cbz), methyl carbamate, ethyl carbamate,9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethylcarbamate, 17-tetrabenzol[a,c,g,i]fluorenylmethyl carbamate (Tbfmoc),2-chloro-3-indenylmethyl carbamate (Climoc),2,7-di-t-butyl[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 1,1-dioxobenzo[b]thiophene-2-ylmethyl carbamate(Bsmoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethylcarbamate (Teoc), 2-phenylethyl carbamate (hZ), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-boc),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBoc),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc),N-2-pivaloylamino)-1,1-dimethylethyl carbamate,2-[(2-nitrophenyl)dithio]-1-phenylethyl carbamate (NpSSPeoc),2-(N,N-dicycloheylcarboxamido)ethyl carbamate, 1-adamanyl carbamate(1-Adoc), cinyl carbamate (Voc), 1-isopropylallyl carbamate (Ipaoc),4-nicrocinnamyl carbamate (Noc), 3-(3′pyridyl)prop-2-enyl carbamate(Paloc), 8-quinolyl carbamate, alkyldithio carbamate, p-methoxybenzylcarbamate (Moz), p-nitrobenzyl carbamate (Pnz), p-bromobenzyl carbamate,p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate,4-methylsulfinylbenzyl carbamate (Msz), diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 1,1-dimethyl-2-cyanoethyl carbamate, 2-(4-nitrophenyl)ethylcarbamate, 4-phenylacetoxybenzyl carbamate (PhAcOZ), andm-chloro-p-acyloxybenzyl carbamate. In some embodiments, the carbamateprotecting group is chosen from 9-fluorenylmethyl carbamate (Fmoc),t-butyl carbamate (Boc), and carboxybenzyl carbamate (cbz).

The amide protecting group may include, for example, acetamide,phenylacetamide, 3-phenylpropanamide, pent-4-enamide, picolinamide,3-pyridylcarboxamide, benzamide, p-phenylbenzamide,2-methyl-2-(o-phenylazophenoxy)propanamide), 4-chlorobutanamide,acetoacetamide, 3-(p-hydroxyphenyl)propanamide), and(N′-dithiobenzyloxycarbonylamino)acetamide.

Examples of suitable protecting groups also include tert-butyl, benzyl,4-methoxybenzyl, benzyloxymethyl, phenacyl, allyl, trimethylsilyl,benzyloxycarbonyl, tert-butoxycarbonyl, and acetal and ketalderivatives. In some embodiments, R is selected from trityls,substituted trityls (e.g., monomethoxytrityl (MMTr), dimethoxytrityl(DMTr), trimethoxytrityl (TMTr), 2-chlorotrityl (ClTr) andp-bromophenacyloxytrityl (BPTr), pixyls and substituted pixyls (see, forexample, U.S. Publication No. 2007/0276139). In some embodiments, R isselected from trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, andcyclic acetal.

In some embodiments, R is a hydrophobic separation handle.

A hydrophobic separation handle is as described herein. In someembodiments, the hydrophobic separation handle is also a protectinggroup as described herein. In some embodiments, at least one of R and Gis a hydrophobic separation handle.

In some embodiments, only one of R and G is a hydrophobic separationhandle (e.g., a trityl group) as provided herein. For example, if R is ahydrophobic separation handle, then G is hydrogen or an alkoxy. In someembodiments, R is a protecting group and G is hydrogen or an alkoxy. Insome embodiments, R is absent and G is a trityloxy group. In someembodiments, the hydrophobic separation handle is a substituted orunsubstituted trityl or trityloxy group. For example, only one of R andG is a substituted or unsubstituted trityl or trityloxy group.

In some embodiments, R² is absent or is selected from the groupconsisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,alkoxypixyl, fluorenylmethyloxycarbonyl, alkylcarboxyl, benzoyl,tetrahydropyranyl, and methyl.

A compound of formula (3) can be prepared, for example, by contacting amonosubstituted polymer comprising a linear, water-soluble, non-peptidicand non-nucleotidic polymer backbone bonded at the first terminus withthe functional group M²-R², with a reagent of formula (5):

wherein:

R⁵ and R⁶ independently from each other represent C₁-C₆-alkyl or R⁵ andR⁶ jointly form a 5- or 6-membered ring with the N to which they arebonded; under conditions facilitating the conversion of themonosubstituted polymer to a compound of formula (3).

In some embodiments, R⁵ and R⁶ are independently a C₁-C₆-alkyl. Forexample, R⁵ and R⁶ can be independently selected from the groupconsisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, and hexyl. In some embodiments, R⁵ and R⁶ areisopropyl. In some embodiments, R⁵ and R⁶ jointly form a 5- or6-membered ring with the N to which they are bonded. For example, R⁵ andR⁶ jointly form a pyrrolidine, pyrroline, imidazoline, pyrazolidine,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyridyl, pyrazinyl, pyrimidinyl,particularly 2- and 4-pyrimidinyl, pyridazinyl, pyrrolyl, particularly2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, and pyrazolyl, particularly3- and 5-pyrazolyl. In some embodiments, R⁵ and R⁶ jointly form amorpholine ring.

The ratio of a monosubstituted polymer to a reagent of formula (5) canrange from about 1:10 to about 10:1 (e.g., about 2:1, about 3:1, about4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 1:2,about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:81 about1:9, about 2:8, about 3:7, about 4:6 about 5:10, and about 4:8). In someembodiments, the ratio of a polymer to a reagent of formula (5) is fromabout 1:1 to about 1:10. In some embodiments, the ratio of a polymer toa reagent of formula (5) is about 2:1.

In some embodiments, the conversion of a monosubstituted polymer to acompound of formula (3) is quantitative.

In some embodiments, conditions that facilitate formation of a compoundof formula (3) include the addition of an activating reagent. Anactivating reagent is then added to the mixture of the monoderivatizedproduct and the reagent of formula (5) or (6). An activating reagent canbe any group suitable to initiate coupling of the polymer and thereagent of formula (5) or (6). Suitable activating reagents include, forexample, 1H-tetrazole, 5-(ethylthio)-1H-tetrazole (ETT),5-(benzylthio)-1H-tetrazole (BTT), Activator 42(5-(3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole), 2-ethylthiotetrazole,2-bezylthiotetrazole, 4,5-dicyanoimidazole and 4,5-dicyanoimidazole(DCI). In some embodiments, an activating agent can be selected frompyridinium hydrochloride, pyridinium trifluoroacetage, and bufferedcarboxylic acids.

In some embodiments, conditions that facilitate formation of a compoundof formula (3) include addition of an oxidizing agent to oxidize P⁺³ toP⁺⁵. Suitable oxidizing agents and conditions can be readily determinedby those of ordinary skill in the art. For example, an oxidant such asRuO₄ ⁻/NMO, Dess-Martin's reagent, DMSO/triflic anhydride, PDC, hydrogenperoxide, inorganic peroxides, nitric acid, nitrates, chlorite,chlorate, perchlorate, hypochlorite, peroxide, iodine, ozone, nitrousoxide, silver oxide, permanganate salts, hexavalent chromium compounds,chromic acid, dichromic acids, chromium trioxide, pyridiniumchlorochromate, persulfuric acid, sulfoxides, sulfuric acid, Tollens'reagent, 2,2′-dipyridiyldisulfide (DPS), and osmium tetroxide may beused.

In some embodiments, iodine can be used as an oxidizing agent. Forexample, a solution of iodine can be used and prepared by dissolvingiodine in a mixture of pyridine, tetrahydrofuran and water. Elementalsulfur can be used for phosphite oxidation combined with formation ofsulfurized product. In some embodiments, other more soluble and morereactive reagents, such as 3H-1,2-benzothiazol-3-one 1,1-dioxide(Beaucage reagent), phenylacetyl disulfide (PADS) or dimethylthiuram(DTD) can be used. Alternatively, peroxides exemplified by t-butylhydrogen peroxide or m-chlorobenzoyl peroxide may be used for P⁺³ to P⁺⁵oxidations.

In some embodiments, an oxidizing reagent is selected from a groupconsisting of: iodine, hydrogen peroxide, t-butyl hydrogen peroxide,acetone peroxide, sulfur, and thiuram disulfide.

In some embodiments, R and/or R¹ is a protecting group or a hydrophobicseparation handle. In some embodiments, the method can further includepurifying the monoderivatized compound using chromatography (e.g.,reverse phase chromatography). In some embodiments, the method furtherincludes removal of one or more of the protecting groups. In someembodiments, the method further includes removal of one or more of thehydrophobic separation handles.

For the methods provided above, the deprotection may involve, forexample, either sequential or one-pot deprotection of certain protectinggroups. Suitable reagents and conditions for the deprotection can bereadily determined by those of ordinary skill in the art. For example,deprotection may be achieved upon treatment of the protected compoundunder conditions so that hydroxyl protecting groups, such as acetate,isopropylidine, benzylidine, trityl, and pixyl protecting groups, areremoved from the protected compound. The acetate group can be cleavedunder mild conditions, for example, by diluted solution of ammonia or bysolution of potassium carbonate. The benzylidene and isopropylidenegroups can be cleaved by hydrogenation or using acidic hydrolysis asreported by R. M. Hann et al., J. Am. Chem. Soc., 72, 561 (1950). In yetanother example, the deprotection can be performed so that aminoprotecting groups, such as 9-fluorenylmethyl carbamate (Fmoc), t-butylcarbamate (Boc), and carboxybenzyl carbamate (cbz) protecting groups arecleaved from the protected compound. 9-fluorenylmethyl carbamate (FMOC)can be removed under mild conditions with an amine base (e.g.,piperidine) to afford the free amine and dibenzofulvene, as described byE. Atherton et al., “The Fluorenylmethoxycarbonyl Amino ProtectingGroup,” in The Peptides, S. Udenfriend and J. Meienhofer, AcademicPress, New York, 1987, p. 1. t-butyl carbamate (Boc) can be removed, asreported by G. L. Stahl et al., J. Org. Chem., 43, 2285 (1978), underacidic conditions (e.g., 3 M HCl in EtOAc). Hydrogenation can be used tocleave the carboxybenzyl carbamate (cbz) protecting group as describedby J. Meienhofer et al., Tetrahedron Lett., 29, 2983 (1988).

In some embodiments, deprotection may be performed under anaerobicconditions. The deprotection may also be performed at ambienttemperature or at temperatures of from about 20-60° C. (e.g., 25, 30,35, 40, 45, 50, or 55° C.).

In some embodiments, the method can also include isolating the compoundof formula (3) by precipitation or crystallization.

Non-limiting examples of linking groups for use in the compoundsprovided herein include:

Provided herein is a new type of functional, water-soluble polymer, notbelonging to the classes of polypeptides or nucleotides and containingthe structure of formula (1) that can be conjugated to a biologicallyactive molecule or a derivative thereof:

In this schematic picture of such a modified polymer, A is the point ofbonding to the terminus of the polymer backbone, E is an oxygen orsulfur atom, K is selected from the group consisting of alkylene,alkyleneoxyalkylene, or an oligomeric form of alkyleneoxyalkylene, G ishydrogen or is selected from the group consisting of an alkoxy and ahydrophobic separation handle, Z¹ and Z² can be oxygen or nitrogen, insuch way that both Z¹ and Z² may be oxygen, but when Z¹ is NH then Z² isoxygen, and when Z² is NH then Z¹ is oxygen, L is selected from thegroup consisting of a divalent radical of a nucleoside, linear alkylene,branched alkylene, alkyleneoxyalkylene, oligomeric form ofalkyleneoxyalkylene, arylene, and substituted arylene, M is a protectedgroup that when deprotected is reactive with a biologically activemolecule or a derivative thereof or is a group reactive with abiologically active molecule or a derivative thereof, R is a protectinggroup, activating group, hydrogen or absent. Thus the L-M-R fragment islinked to a terminus of the polymer via a phosphotriester,thiophosphotriester or amidophosphotriester group.

One characteristic of a compound provided herein is that the functionalgroup M, via the group L, is connected to the chain of the polymer via aphosphotriester group or amido phosphodiester, known also asphosphoramidate group. In addition, a derivatized polymer may exist bothin an oxy and a thio form. Non-limiting examples of such groups include:

An important class of polymers provided herein, polyethylene glycols(PEG), were previously used in the synthesis of phosphoramidites of theformula: DMTr-O-PEG-O—P(OCE)N(iPr)₂. These compounds were used fordirect coupling of PEG molecules to synthetic nucleic acids or to asurface of a solid phase. In all reactions, the reactive phosphoramiditegroup was present at the terminal of the polymer. A polymer substitutedwith a phosphoramidite group is, however, not a subject of the presentdisclosure, as the phosphorous atom in the phosphoramidite group is thepart of the reactive functionality and not a part of the linker as inthe compounds provided herein.

This formal distinction can have a deeper chemical importance. Aphosphoramidite group can be designed to work in a completely water-freeenvironment. Upon activation with certain protonating agents/activatorsa phosphoramidite group can become extremely reactive, and in thepresence of water, this reactive function can decompose instantaneously,making this function inappropriate for conjugation to a biologicallyactive molecule or a derivative thereof in aqueous solution.Additionally, published PEG phosphoramidites can contain a speciallydesigned, labile protecting group adjacent to the phosphorous atom toconvert the intermediate phosphotriester to a phosphodiester. Thepresent document is based, at least in part, on the observation thatphosphodiesters are unstable in aqueous biological media such as blood,plasma or cellular extracts, due to the presence of phosphatases andphosphodiesterases. This can preclude phosphodiester linkages as alinking group within a structure of a conjugate, as long as it is aimedfor use in biological media. This document, contrary to the existingliterature, and contrary to all normal procedures, recommends keepingthe phosphate in the form of a phosphotriester or in the form of itsamide in order to gain stability of the linking group/conjugate.

Phosphotriester bonds are very rare in nature, existing in most of thecases as cyclic products of RNA transformations. As non-charged variantsof nucleotides they gained some attention from those who hoped that itcould be possible to use this form of nucleotides as predrugs, whichwould be transformed in vivo to the active phosphodiester forms.However, acyclic phosphotriesters, lacking specially designed internalfeatures facilitating deprotection, were found completely stable, bothchemically at the physiological pH range, and enzymatically in thepresence of the most active phosphate hydrolytic enzymes: McGuigan etal., Nucl. Acids Res. 1989, 17 (15), 6065-6075; Hecker and Erion, J.Med. Chem. 2008, 51, 2328-2345; Conrad et al. Chem. Bio. Interactions1986, 60, 57-65; and Fidanza et al., Methods in Molecular Biology 1994,26, 121-143.

The proposed way of linking a functional group to the polymeric moleculecan include combining chemistry typical for nucleic acids with chemistryof polymers and their conjugates. Moreover, this combination can beperformed in a way to yield a product with distinctly bettercharacteristics than if this combination of chemistries would proceedfollowing the standard path.

This document also provides methods and materials for introducing of auseful separation handle on the derivatized polymer. This separationhandle can be introduced simultaneously with the functional group, sothe presence of the separation handle becomes an indicator of successfulintroduction of the reactive group. If chromatographic properties of aparticular separation handle are properly chosen, it is possible todiscriminate between non-derivatized, monoderivatized andmultiderivatized (e.g., bis-derivatized) polymers. Most of theseparation handles used herein introduce hydrophobic properties to thepolymer, as the preferred method for separation of the modified polymersis based on reverse-phase analytical and preparative chromatography. Thechoice of a proper separation handle comes from consideration of severalpractical aspects such as:

a) The hydrophobic separation handle can be removed from the polymerafter purification in order to liberate the group reactive with abiologically active molecule and to avoid uncontrolled hydrophobicinteractions within the conjugate—or more generally to avoiduncontrolled hydrophobic interactions during the interaction of theconjugate with the biological environment. This can preclude work withanalogues of mPEG which contain a long hydrophobic alkyl ether chaininstead of a methoxy group at one of the polymer termini. For the samereason, protection of an amino group as long chain fatty acid amides isnot practical, as this group can be removed only under very extremeconditions.

b) As most of polymers and functional groups lack chromophoricproperties, the chromatographic separation of polymers can be difficult.It is, therefore, advantageous if the separation handle introducesadditionally some chromophore properties to the polymer. This can makeprotection of a terminal hydroxyl or thiol group by means of a longchain aliphatic fatty acid ester less interesting.

c) Chemical stability of the hydrophobic separation handle can be easyto control depending on an actual situation. This aspect is relatedmostly to the stability of other functional groups present in thederivatized polymer.

d) Chromatographic properties of the hydrophobic separation handle, andhence the properties of the derivatized polymer, can be easy to controlby chemical modification of the hydrophobic separation handle.

e) Since even relatively stable phosphotriester bonds are slightlylabile under high pH conditions, it can be preferred to avoidhydrophobic separation handles that can only be removed under suchconditions.

Examples of hydrophobic separation handles that fulfill all thesecriteria belong to the group of acid labile protecting groups and areknown as trityls, substituted trityls (e.g., monomethoxytrityl (MMTr),dimethoxytrityl (DMTr), trimethoxytrityl (TMTr), andp-bromophenacyloxytrityl (BPTr), pixyls and substituted pixyls (see, forexample, U.S. Publication No. 2007/0276139). They are all acid labile,with distinct UV chromophore properties. Their acid stability can beeasily controlled by the presence of different electron donating orelectron withdrawing groups. Introduction of alkoxy chains of differentlength to the trityl structure is a convenient method for modificationof their hydrophobic properties. Most of the Examples presented hereinutilize, therefore, trityl groups both for protection of reactivefunctions and for introduction of an efficient separation handle. Thisshould not be seen as a limitation of this disclosure, as other groups,even those which do not fulfill all the above criteria, may be useful inthe present methodology as hydrophobic separation handles. Thus, ageneral description of a potential hydrophobic separation handleprovided herein is: a hydrophobic group that withstands the presence oftrivalent phosphorus present in a phosphoramidite or in an activatedH-phosphonate, and which can also be employed for chromatographicresolution of modified polymer from the unmodified starting material.

With a few exceptions, there are few limits on the type and character ofa functional group M. These exceptions appear in cases when thefunctional group is very sensitive to reducing conditions and becomesdestroyed by the trivalent phosphoramidite group, with concomitantoxidation of phosphorus to the pentavalent oxidation state. The azidogroup is an example of such a reactive function that cannot be convertedto the appropriate phosphoramidite. In fact, trivalent phosphorus oftriphenylphosphine is used as an efficient reagent for conversion of anazido group to an amine. The activated dithio group, for example, as ina dithiopyridyl group, is another example that belongs to this category,although simple dithiols could be successfully converted to anddelivered in the form of a phosphoramidites. Nevertheless, thisdisclosure presents also a variant of the above phosphoramidite methodthat omits the mentioned stability problem, and provides the ability toprepare polymers containing even an azido or activated dithio group.

This method is known as H-phosphonate methodology followed by oxidationof P⁺³ by carbon tetrachloride/amine, and is similar to thephosphoramidite method in the sense that the incoming reagent containsreactive P⁺³ phosphorus, and the phosphorus atom is oxidized during thereaction process to its pentavalent stage. The H-phosphonate methodologywill be described in more detail later on in this text.

Many groups reactive with a biologically active molecule or a derivativethereof need to be protected in order to exist within the structure ofphosphoramidite reagent. Examples include, without limitation, amino,aminoxy, hydrazo, hydroxyl, thio, certain fluorophores and carboxygroups. Some of these groups, like biotin, do not demand protection, butcan be used in a protected form to obtain some additional effects. Insome cases, trityl, substituted trityl, pixyl, and substituted pixyl canbe used as protecting groups. One reason for this choice of a protectinggroup is the possibility for simultaneous introduction of a protectinggroup that also can be used as a hydrophobic separation handle in areverse phase (RP) based chromatographic separation process. Ifseparation is not demanded, like in the case where the polymer has onlyone reactive terminus (e.g., when using mPEG as a polymer backbone), andincorporation of a phosphoramidite may be forced to completion, anyprotecting group can be used for protection of the group reactive with abiologically active molecule. In particular, trifluoroacetamido and FMOCgroups may be used for the protection of amino, aminoxy and hydrazogroups in such phosphoramidites.

The use of phosphoramidites containing a protected group, that whenunprotected is reactive with a biologically active molecule or aderivative thereof, having a hydrophobic protecting group forderivatization of polymers has a clear advantage over other methods. Forexample, the starting polymer does not have to be partially protected inorder to obtain pure, monofunctionalized polymers. The methods work withfully unblocked polymers and improved yield is obtained using an excessof such a non-expensive polymer (e.g., non-derivatized PEG) over theamidite. One value of this method is in the fact that the formed mixtureconsisting of mono-, bis-, or multi-derivatized polymers can beefficiently separated from each other.

Certain functional groups do not offer a straightforward possibility forintroduction of a desired hydrophobic protecting group. To this groupbelong NHS-esters, most of the fluorophores, iodoacetamido and maleimidogroups. Amidites containing these functional groups are not optimal forderivatization of diol-polymers, since the separation of the reactionmixtures can be impossible.

In some cases, polymer diols can be easily converted to certainmonoprotected derivatives, and this protection can offer the possibilityfor using chromatography to obtain pure, temporarily blockedmonoderivatized polymers. Even here the separation can be based onhydrophobic interactions between a hydrophobic support and a polymerderivatized with a hydrophobic protecting group acting as a hydrophobicseparation handle. Examples of handles used for this purpose include,without limitation, substituted or unsubstituted tritylated hydroxyls,tritylated thiols, and tritylated amines. Introduction of a trityloxy orpixyloxy group onto the terminus of a polymer is straightforward asthese polymers usually contain free hydroxyl groups. Introduction of atritylthio group requires activation of the hydroxyl group by itsconversion to a mesylate, tosylate, or by substitution with a halogen.In some cases, this last alternative can be used, as its implementationin the form of a Velsmeier reaction can be economically attractive. Theactivated polymer can then react with tritylmercaptan as described byConolly and Rider in Nucleic Acids Res. 1985, 13, (12), 4485-4502. Apolymer substituted with a thiol group also can be obtained by any ofthe other described methodologies, and then the thio group can beselectively tritylated in an acidic environment, utilizing the muchhigher affinity of the thio group over hydroxyl to carbocations. Thereare two ways to obtain polymers protected on one site with a tritylatedamine group. A process is described for direct alkylation of tritylaminewith alkyl halides so this method could be used directly in analogy tothe above alkylation of tritylmercaptan. On the other hand anyappropriate method for partial amination like alkylation of thephthalimide, alkylation of the trifluoracetamide or a Mitsunobuprocedure can be applied for preparation of a monoamino polymer. Thisstarting material, even in unpurified form, can be used for obtainingthe tritylated amino polymer in a two-step process, starting withsilylation of all free hydroxyls in a pyridine solution. All mentionedtritylated or pixylated polymers can be preoperatively purified by RPchromatography to isolate the pure monosubstituted polymer. Thedescribed earlier mono-protected polymer derivatives, made by reacting apolymer with a selected phosphoramidite, offer an interestingalternative as a mono-protected polymeric starting material foradditional derivatization. Such a polymer can react next with aphosphoramidite to incorporate another functional group, so the finalproduct can contain, for example, two separate phosphotriester linkages.

Monomethoxy PEG (mPEG), or generally monoalkylated polymers, can be usedfor the same purpose. Methoxy PEG's are used to guaranteemonofunctionalization of a polymer. The problem is that such polymerscan be contaminated by an unknown and variable amount of bis hydroxyl(diol) polymer. In the case of PEG the existence of the diol form is aconsequence of moisture present during the polymerization of theethyleneoxide, and this can be hard to avoid on an industrial scale.Even the slightest amount of water can result in formation of a hydroxylanion—an undesired starting point of polymerization. Thus mPEG is not anoptimal polymer for preparation of pure monofunctionalized polymers.

Once a polymer is properly monoprotected, however, its conversion to areactive monofunctional derivative is straight-forward. Phosphoramiditechemistry allows incorporation of a reactive functionality with yieldand speed that is beyond the competition of other chemical processes,including all sorts of hydroxyl alkylation reactions. Using the properexcess of these reagents, the reported yields exceed 98% for every stepin a multistep process and are often nearly quantitative. In cases whereonly a single incorporation of a phosphoramidite takes place, thisreaction can be expected to be quantitative.

There exists also an interesting alternative process to the describedabove functionalization following the previously describedmonoprotection. Groups reactive with a biologically active molecule or aderivative thereof that lack the possibility of carrying a hydrophobicseparation handle can be incorporated into a trisubstituted unit havingthe reactive group of choice, the hydrophobic separation handle and thephosphoramidite group. There are several such units or molecules thatcan be used as carriers (scaffolds) for the construction of suchtrisubstituted block reagents. One of the simplest molecules, due to theease of the chemical manipulations, is the uridine-based scaffolddescribed by Hovinen and Hakala in Organic Lett. 2001, 3(16) 2473-2476,where R denotes a reactive group and L is an aliphatic linker to theuridine ring.

For instance, using reagents belonging to this category, it is possibleto prepare, in a single reaction step, polymers covalently bonded todifferent fluorescent functionalities that can be isolated as amonosubstituted polymeric product.

The chemistry of phosphoramidites is mostly associated with thechemistry of nucleic acids. The very high demands of this multistepprocess require rather large excess of incoming amidites, and efficientcatalysts. Examples of activators includes, without limitation,1H-tetrazole, 5-(ethylthio)-1H-tetrazole (ETT),5-(benzylthio)-1H-tetrazole (BTT), Activator 42(5-(3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole), and4,5-dicyanoimidazole (DCI). Those are often expensive, and if applied tothe synthesis of polymers, as in this disclosure, they would noticeablyincrease the price of the final reagent. However, as the chemistry inthe present disclosure can have a single coupling step, and thequantitative yield of the reaction is not always necessary, it ispossible to use other less expensive catalysts. The use of pyridiniumhydrochloride or even buffered carboxylic acids for activation ofphosphoramidites is described and proved, offering high reaction rate,albeit with minimally lower coupling yield.

The choice of an oxidizing reagent is another factor for consideration.Iodine is the simplest alternative, as this reagent can be prepared bydissolving iodine in a mixture of pyridine, tetrahydrofuran and water.Elemental sulfur can be used for phosphite oxidation combined withformation of sulfurized product, but it can be poorly soluble in organicsolvents which can be used in the procedures described herein. Other,better soluble and more reactive reagents, like3H-1,2-benzothiazol-3-one 1,1-dioxide (Beaucage reagent), phenylacetyldisulfide (PADS) or dimethylthiuram (DTD) were developed for thispurpose. Thiophosphotriester linkages do not offer very much advantageover the normal phosphotriester linkages, as the latter can already besufficiently stable, but may offer additional possibilities, e.g. theability of chemical cleavage of this linkage. Peroxides exemplified byt-butyl hydrogen peroxide or m-chlorobenzoyl peroxide can be used asalternatives for P⁺³ to P⁺⁵ oxidations. They are colorless, can workunder water-free conditions and are often applicable in situations whereiodine promoted oxidations may lead to some unwanted side reactions.

It was mentioned earlier that groups reactive with a biologically activemolecule which do not tolerate a coexistence with the phosphoramiditegroup can be attached to the polymer by H-phosphonate chemistry. Thismethod takes advantage of the fact that P⁺³ of the H-phosphonate isactually tetracoordinated, and as such this group can be more stable foroxidation than a phosphate triester or phosphoramidite. The particularlyimportant point is that H-phosphonate does not interact with an azidogroup like most P⁺³ containing compounds. The use of this methodologyfor incorporation of the azido group can be, however, combined with theincorporation of a useful hydrophobic separation handle allowing fordiscrimination between the product and unreacted starting material.Application of a trifunctional reagent, similar to the mentioned uridinederivative, but using a H-phosphonate instead of a phosphoramiditegroup, could be an alternative. In this case, the activation of thestarting H-phosphonate reagent and its reaction with the polymer can befollowed by addition of an excess of an appropriate alcohol (e.g.,isopropanol) to convert the formed H-phosphonate diester to the desiredH-phosphonate triester. The Examples below describe a slightly differentprocedure. Here, the procedure of introducing an azido group and ahydrophobic separation handle has been divided into two steps, insteadof making it in a single step, using a trifunctional reagent. This isnot an optimal approach, but it was possible due to very high yield ofeach reaction. Polymer was first reacted with H-phosphonate oftritylated diethyleneglycol. It is important to use a starting glycolcontaining more than two carbons; otherwise, the free OH group in thefinal product, after removal of the trityl group, may destabilize thephosphotriester linkage. Oxidation of P⁺³ to P+5 was combined with theintroduction of the azido group into the polymer, and it was doneaccording to the described CCl₄/pyridine/amine procedure, using1-amino-6-azidohexane as a source of the azido group. The opposite orderof incorporation of reactive groups using H-phosphonate chemistry isalso feasible. It is also recognized that H-phosphonate chemistry couldbe applied for the incorporation of any of existing reactive groups, ifother factors of interest like hydrolytic stability of the startingreagents, or costs of syntheses, are deciding.

Conjugates

This document also provides conjugates that include a functionalizedpolymer as provided herein and a biologically active molecule.

In some embodiments, a biologically active molecule is a TNF inhibitoror a derivative thereof. As used herein, the term “TNF inhibitor”includes antibodies or fusion proteins that bind to TNF alpha (e.g.,human TNF alpha). Non-limiting examples of include etanercept (Enbrel®,Amgen and Pfizer); infliximab (Remicade®, Janssen Biotech, Inc.);adalimumab (Humira®, Abbott Laboratories); certolizumab pegol (Cimzia®);and Golimumab (Simponi®, Janssen Biotech, Inc.).

In some embodiments, a biologically active molecules is insulin or aderivative thereof. Derivatives of insulin include, for example, insulinanalogs that contain one or more amino acid substitutions, andmodifications to insulin or insulin analogs to include, for example, amoiety reactive with M or R on the polymer (as described above forcompounds (1), (2), and (3)). Insulin and analogs can be recombinantlyproduced (e.g., in a non-pathogenic organism). Non-limiting examples ofsuitable insulin analogs include insulin lispro (HUMALOG, sold by EliLilly), insulin aspart (“NovoLog/NovoRapid” sold by Novo Nordisk),insulin detemir (Levemir® sold by Novo Nordisk), Insulin glargine(Lantus®, sold by Sanofi-Aventis), and insulin glulisine (Apidra®, soldby Sanofi-Aventis).

Insulin lispro is a rapid-acting human insulin analog used to lowerblood glucose levels, and contains a substitution of lysine for prolineat position B28, and a substitution of proline for lysine at positionB29.

Insulin aspart is a rapid-acting human insulin analog used to lowerblood glucose levels, and contains a substitution of an aspartic acidfor proline at position B28.

Insulin detemir is a long-acting insulin analog used for maintaining thebasal level of insulin. Insulin determir contains myristic acid is boundto the lysine at position B29.

Insulin glargine is a long-acting insulin analog used for lower bloodglucose, and contains a substitution of a glycine for asparagine atposition A21 and contains two arginines added to the C-terminus of theB-chain.

Insulin glulisine is a rapid acting human insulin analog used to lowerblood glucose levels, and contains a substitution of a lysine forasparagine at position B3 and the substitution of a glutamic acid forlysine at position B29.

In some embodiments, a biologically active molecule is omalizumab or aderivative thereof. Omalizumab is sold under the trade name Xolair(Novartis Pharmaceuticals, East Hanover, N.J.; Genentech Inc., South SanFrancisco, Calif.), and is a recombinant humanized monoclonal antibodyhaving a molecular weight of approximately 150,000 Da. See SubstanceIdentifier 47206967. See also U.S. Pat. No. 6,267,958.

“Antibody” as the term is used herein refers to a protein that generallycomprises heavy chain polypeptides and light chain polypeptides. Antigenrecognition and binding occurs within the variable regions of the heavyand light chains. Single domain antibodies having one heavy chain andone light chain and heavy chain antibodies devoid of light chains arealso known. A given antibody comprises one of five types of heavychains, called alpha, delta, epsilon, gamma and mu, the categorizationof which is based on the amino acid sequence of the heavy chain constantregion. These different types of heavy chains give rise to five classesof 15 antibodies, IgA (including IgA1 and IgA2), IgD, IgE, IgG (IgG1,IgG2, IgG3 and IgG4) and IgM, respectively. A given antibody alsocomprises one of two types of light chains, called kappa or lambda, thecategorization of which is based on the amino acid sequence of the lightchain constant domains. Omalizumab is an IgG1 antibody and containskappa chains. IgG antibodies generally contain two identical heavychains and two identical light chains and two antigen combining domains,each composed of a heavy chain variable region (V_(H)) and a light chainvariable region (V_(L)).

“Humanized antibody” refers to an antibody that has been engineered tocomprise one or more human framework regions in the variable regiontogether with non-human (e.g., mouse, rat, or hamster)complementarity-determining regions (CDRs) of the heavy and/or lightchain. In some embodiments, a humanized antibody comprises sequencesthat are entirely human except for the CDR regions. Humanized antibodiesare typically less immunogenic to humans, relative to non-humanizedantibodies, and thus offer therapeutic benefits in certain situations.For Omalizumab, the CDRs are derived from the parent murine monoclonalantibody and the constant regions of Omalizumab are derived from IgG₁.

Derivatives of Omalizumab include, for example, modifications toOmalizuabinsulin or insulin analogs to include, for example, a moietyreactive with M or R on the polymer (as described above for compounds(1), (2), and (3)) or a fragment of Omalizumab. A fragment of Omalizumabrefers to a polypeptide derived from the heavy or light chain ofOmalizumab that lacks all of part of at least one chain of Omalizumab.As the term as used herein encompasses fragments that comprise singlepolypeptide chains derived from antibody polypeptides (e.g., a heavy orlight chain antibody polypeptide), it will be understood that anantibody fragment may not, on its own, bind an antigen. For example, anantibody fragment may comprise that portion of a heavy chain antibodypolypeptide that would be contained in a Fab fragment; such an antibodyfragment most commonly will not bind an antigen unless it associateswith another antibody fragment derived from a light chain antibodypolypeptide (e.g., that portion of a light chain antibody polypeptidethat would be contained in a Fab fragment) and reconstitutes theantigen-binding site. Non-limiting examples of antibody fragments caninclude, for example, polypeptides that would be contained in Fabfragments, F(ab′)2 fragments, or scFv (single chain Fv) fragments.

In some embodiments, a biologically active molecule is a clotting factoror a derivative thereof. Derivatives of a clotting factor include, forexample, a clotting factor analog that contains one or more amino acidsubstitutions relative to the corresponding human clotting factor, andmodifications to the clotting factor or clotting factor analog toinclude, for example, a moiety reactive with M or R on the polymer (asdescribed above for compounds (1), (2), and (3)). Clotting factors andanalog thereof s can be recombinantly produced (e.g., in anon-pathogenic organism).

In some embodiments, a polypeptide that boosts red or white blood cellproduction. Examples of polypeptides that boost red blood cellproduction include epoetin alpha (Epogen®, Amgen), epoetin beta(NeoRecormon®, Roche), epoetin theta (Eporatio®), epoetin zeta anddarbepoetin alpha (Aranesp®, Amgen)). Examples of polypeptides thatboost white blood cell production include filgrastim (e.g., Neupogen®).Such polypeptides can be recombinantly produced. Derivatives of apolypeptide that boosts red or white blood cell production include, forexample, an analog that contains one or more amino acid substitutionsrelative to the corresponding human polypeptide, and modifications tothe polypeptide or analog to include, for example, a moiety reactivewith M or R on the polymer (as described above for compounds (1), (2),and (3)).

This document also provides conjugates that include a functionalizedpolymer as provided herein and a biologically active molecule such as anantibody or a derivative thereof. Non-limiting examples of antibodiesthat can be conjugated to a functionalized polymer include, abatacept(Orencia®), alemtuzumab (marketed as Campath, MabCampath or Campath-1H);basiliximab (Simulect®), belimumab (Benlysta®), besilesomab(Scintimun®), bevacizumab (Avastin®), canakinumab (Ilaris®), catumaxomab(Removab®), cetuximab (Erbitux®), denosumab (Prolia®, Xgeva®),eculizumab (Soliris®), ipilimumab (also known as MDX-010 or MDX-101,Yervoy®), natalizumab (Tysabri®), ofatumumab (Arzerra®), palivizumab(Synagis®), panitumumab (Vectibix®), ranibizumab (Lucentis®), rituximab(Rituxan®, MabThera®), tocilizumab (Actemra®, RoActemra®), trastuzumab(Herceptin®), and ustekinumab (Stelara®).

Abatacept is sold under the trade name Orencia® by Bristol˜Myers SquibbCompany, and is a fusion protein of the Fc region of IgG1 fused to theextracellular domain of CTLA-4. It is a selective T cell costimulationmodulator indicated for the treatment of adult rheumatoid arthritis andfor juvenile idiopathic arthritis (JIA) in pediatric patients six yearsof age and older.

Alemtuzumab is sold under the trade name Campath®, MabCampath®, orCampath-1H® by Genzyme. Alemtuzumab is a recombinant DNA-derivedhumanized monoclonal antibody that is directed against the 21-28 kD cellsurface glycoprotein, CD52. It is an IgG1 kappa antibody with humanvariable framework and constant regions, and complementarity-determiningregions from a murine (rat) monoclonal antibody (Campath-1G). Campath isproduced in mammalian cell (Chinese hamster ovary) suspension culture ina medium containing neomycin, and is used in the treatment of chroniclymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL) and T-celllymphoma. It is also used in some conditioning regimens for bone marrowtransplantation, kidney transplantation and islet cell transplantation.

Basiliximab is sold under the trade name Simulect® by NovartisPharmaceuticals. It is a chimeric mouse-human monoclonal antibody to thea chain (CD25) of the IL-2 receptor of T cells. It is used to preventrejection in organ transplantation, especially in kidney transplants.

Belimumab is sold under the trade name Benlysta® (Human Genome Sciences,Inc. and The GlaxoSmithKline Group of Companies). It is a monoclonalantibody that inhibits Beta cell activating factor (BAFF), and is usedto treat adults with active systemic lupus erythematosus (SLE or lupus).

Besilesomab is sold under the trade name Scintimun® (CiS BioInternational). It is a mouse monoclonal antibody labeled with theradioactive isotope technetium-99m. It is used to detect inflammatorylesions and metastases. It binds to IgG₁.

Bevacizumab is sold under the trade name Avastin® by Genentech/Roche. Itis a humanized monoclonal antibody that inhibits VEGF-A and slowsangiogenesis. It is used to treat various cancers, including colorectal,lung, breast, glioblastoma, kidney, and ovarian cancer.

Canakinumab is sold under the trade name Ilaris® by Novartis. It is ahuman monoclonal antibody targeted at interleukin-1 beta. It has nocross-reactivity with other members of the interleukin-1 family,including interleukin-1 alpha. Canakinumab is used to treatautoinflammatory syndromes including familial cold autoinflammatorysyndrome, Muckle-Wells syndrome, and neonatal-onset multisysteminflammatory disease.

Catumaxomab is sold under the trade name Removab® by TRION Pharma GmbH.It is a rat-mouse hybrid monoclonal antibody that is used to treatmalignant ascites, which typically occurs in patients with metastasizingcancer. Catumaxomab has two binding specificities directed at epithelialcell adhesion molecule (EpCAM) and the T-cell antigen CD3. See Sebastianet al., Drugs Today (Barc). 45(8):589-97 (2009).

Cetuximab is sold under the trade name Erbitux® by Bristol-Myers Squibband Eli Lilly and Company. It is a chimeric (mouse/human) monoclonalantibody that binds to the epidermal growth factor receptor (EGFR) andacts as an inhibitor. Cetuximab can be used for treatment of metastaticcolorectal cancer and head and neck cancer.

Denosumab is sold under the trade name Prolia® and Xgeva® by Amgen. Itis a fully human monoclonal antibody that targets RANKL (RANK ligand), aprotein that acts as the primary signal for bone removal, and is usedfor the treatment of osteoporosis, treatment-induced bone loss, bonemetastases, rheumatoid arthritis, multiple myeloma, and giant cell tumorof bone.

Eculizumab is sold under the trade name Soliris® by AlexionPharmaceuticals. It is a recombinant humanized monoclonal antibody thatcontains human constant regions from human IgG₂ sequences and human IgG₄sequences and murine complementarity-determining regions grafted ontothe human framework light- and heavy-chain variable regions. Eculizumabis composed of two 448 amino acid heavy chains and two 214 amino acidlight chains and has a molecular weight of approximately 148 kDa.Eculizumab specifically binds the complement protein C5. Eculizumab hasbeen shown to be effective in treating paroxysmal nocturnalhemoglobinuria (PNH), a rare and sometimes life threatening disease ofthe blood. Binding of Eculizumab to C5 inhibits its cleavage by the C5convertase, and prevents the generation of the terminal complementcomplex C5b-9. It is this terminal complement complex that causesintravascular hemolysis in people with PNH.

Ipilimumab (also known as MDX-010 or MDX-101) is sold under the tradename Yervoy® by Bristol-Myers Squibb. It is a fully human monoclonalantibody that binds to CTLA-4 (cytotoxic T lymphocyte-associated antigen4), and is used to treat melanoma (e.g., metastatic melanoma orunresectable melanoma).

Natalizumab is sold under the trade name Tysabri® by Biogen Idec andÉlan. It is a humanized monoclonal antibody against the cell adhesionmolecule α4-integrin. Natalizumab is used in the treatment of multiplesclerosis and Crohn's disease.

Ofatumumab is sold under the trade name Arzerra® by Genmab. It is ahuman monoclonal antibody that specifically binds the CD20 protein andinhibits early-stage B lymphocyte activation. Ofatumumab is used fortreating chronic lymphocytic leukemia, follicular non-Hodgkin'slymphoma, diffuse large B cell lymphoma, rheumatoid arthritis, andrelapsing remitting multiple sclerosis.

Palivizumab is sold under the trade name Synagis® by MedImmune. It is ahumanized monoclonal antibody (IgG₁ kappa) directed against an epitopein the A antigenic site of the F protein of RSV. Palivizumab is acomposite of human (95%) and murine (5%) antibody sequences.

Panitumumab is sold under the trade name Vectibix® by Amgen. It is afully human monoclonal antibody specific to the epidermal growth factorreceptor (also known as EGF receptor, EGFR, ErbB-1 and HER1 in humans),and is used to treat metastatic colorectal cancer.

Ranibizumab is sold under the trade name Lucentis® by Novartis. It is amonoclonal antibody fragment (Fab) derived from the same parent mouseantibody as bevacizumab and targets VEGF-A. Ranibizumab is ananti-angiogenic and can be used to treat age-related maculardegeneration (e.g., AMD and ARMD).

Rituximab is sold under the trade names Rituxan® and MabThera® by BiogenIdec and Genentech. It is a chimeric monoclonal antibody against theprotein CD20, which is primarily found on the surface of B cells.Rituximab can be used to treat lymphomas, leukemias, transplantrejection and some autoimmune disorders.

Tocilizumab is sold under the trade names Actemra® and RoActemra® byHoffmann-La Roche and Chugai. It is a humanized monoclonal antibody thatbinds the interleukin-6 receptor (IL-6R) and is used as animmunosuppressive to treat rheumatoid arthritis (RA) and systemicjuvenile idiopathic arthritis.

Trastuzumab is sold under the trade name Herceptin® by Genentech. It isa humanized monoclonal antibody that binds domain IV of theextracellular segment of the HER2/neu receptor. It typically is used totreat breast cancer.

Ustekinumab is sold under the trade name Stelara® by Centocor. It ishuman monoclonal antibody that is directed against interleukin 12 andinterleukin 23, and is used to treat psoriasis.

In certain embodiments, “Chimeric antibody” as the term is used hereinrefers to an antibody that has been engineered to comprise a humanconstant region. Chimeric antibodies are typically less immunogenic tohumans, relative to non-chimeric antibodies, and thus offer therapeuticbenefits in certain situations.

Derivatives of an antibody include, for example, modifications to theantibody to include, for example, a moiety reactive with M or R on thepolymer (as described above for compounds (1), (2), and (3)) or afragment of the antibody. A fragment of the antibody refers to apolypeptide derived from the heavy or light chain of the antibody thatlacks all of part of at least one chain of the antibody. As the term asused herein encompasses fragments that comprise single polypeptidechains derived from antibody polypeptides (e.g., a heavy or light chainantibody polypeptide), it will be understood that an antibody fragmentmay not, on its own, bind an antigen. For example, an antibody fragmentmay comprise that portion of a heavy chain antibody polypeptide thatwould be contained in a Fab fragment; such an antibody fragment mostcommonly will not bind an antigen unless it associates with anotherantibody fragment derived from a light chain antibody polypeptide (e.g.,that portion of a light chain antibody polypeptide that would becontained in a Fab fragment) and reconstitutes the antigen-binding site.Non-limiting examples of antibody fragments can include, for example,polypeptides that would be contained in Fab fragments, F(ab′)2fragments, or scFv (single chain Fv) fragments.

This document also provides conjugates that include a functionalizedpolymer as provided herein and a biologically active molecule such astherapeutic agents, small molecules such as synthetic drugs, oligo- andpolypeptides, oligonucleotides, coding DNA sequences, antisense DNAsequences, mRNAs, antisense RNA sequences, RNAIs, and siRNAs,carbohydrates, lipids, growth factors, enzymes, transcription factors,toxins, antigenic peptides (as for vaccines), antibodies, and antibodyfragments. For example, a biologically active molecule can be agalsidasealfa (Replagal®), agalsidase beta (Fabrazyme®), alglucosidase alfa(Myozyme®), anakinra (Kineret®), bortezomib (Velcade®), conestat alfa(Ruconest®), choriogonadotropin alfa (e.g., Ovidrel®), cinacalcet(Sensipar®), Corifollitropin alfa (Elonva®), dibotermin alfa(InductOs®), eltrombopag (Promacta®), eptotermin alfa (Osigraft®),erlotinib (Tarceva®), follitropin alfa/lutropin alfa, follitropin beta,galsulfase (Naglazyme®), Gefitinib (Iressa®), Human normalimmunoglobulin, human normal immunoglobulin (ivig), human normalimmunoglobulin (SCIg), idursulfase (Elaprase®), Imiglucerase(Cerezyme®), interferon alfa-2b, interferon beta-la, interferon beta-1b,Lapatinib (Tykerb®, Tyverb®), Laronidase (Aldurazyme®), Pazopanib(Votrient), pegaptanib, pegfilgrastim, peginterferon alfa-2b,Rasburicase (Elitek®), Reteplase (Retavase®), Somatropin, Sorafenib(Nexavar®), Tenecteplase (TNKase), thyrotropin alfa, Vandetanib(Caprelsa®), and Vemurafenib (Zelboraf®).

Agalsidase alfa and Agalsidase beta (Fabrazyme®) are recombinant humanα-galactosidase A enzymes, and can be used for long-term enzymereplacement therapy in patients with Fabry's disease, which is caused bythe lack of alpha-galactosidase A.

Aglucosidase alfa is recombinant alpha glucoside, and can be used forenzyme replacement therapy in patients with Pompe's disease.

Anakinra (Kineret®) is an interleukin 1 receptor antagonist, and can beused to reduce the pain and swelling associated with rheumatoidarthritis.

Bortezomib (Velcade®) is an N-protected dipeptide used to treat peoplewith multiple myeloma

Conestat alfa is a recombinant human C1 obtained from the milk oftransgenic rabbits. It can be used to treat hereditary angioneuroticedema.

Choriogonadotropin alfa (Ovidrel®) is human chorionic gonadotropin(HCG), and can be used to help produce mature eggs during in vitrofertilization and stimulate ovulation (release of an egg).

Cinacalcet is a calcimimetic agent that increases the sensitivity of thecalcium-sensing receptor to activation by extracellularcalciumcinacalcet. See e.g., U.S. Pat. No. 6,211,244. It can be used totreat secondary hyperparathyroidism.

Corifollitropin alfa (Elonva®) is a modified follicle stimulatinghormone (FSH), and can be used to stimulate egg production duringinfertility treatments.

Dibotermin alfa (InductOs®) is recombinant human bone morphogeneticprotein 2 and can be used to promote new bone formation and for thetreatment of bony defects.

Eltrombopag (Promacta®) is a small molecule agonist of the c-mpl (TpoR)receptor, and is used to increase the number of platelets (cells thathelp the blood clot) in order to decrease the risk of bleeding in peoplewho have chronic idiopathic thrombocytopenic purpura.

Eptotermin alfa (Osigraft®) is recombinant human bone morphogeneticprotein 7 (hrBMP-7) (also referred to as Osteogenic protein-1 (PP-1))and is used to aid healing of bone.

Erlotinib (Tarceva®) is a reversible tyrosine kinase inhibitor that actson the epidermal growth factor receptor (EGFR). It is used for treatmentof nonsmall cell lung cancer and pancreatic cancer (in combination withgemcitabine).

Follitropin alfa/lutropin alfa contains a recombinant folliclestimulating hormone (FSH) and recombinant luteinizing hormone (LH).Follitropin beta is another recombinant FSH.

Galsulfase is a recombinant form of human N-acetylgalactosamine4-sulfatase (Naglazyme®; BioMarin) and is used for treatment ofmucopolysaccharidosis type VI

Gefitinib (Iressa®) is an EGFR inhibitor that is used for treatment ofcancer (e.g., nonsmall cell lung cancer).

Idursulfase (Elaprase®) is the recombinant lysosomal enzymeiduronate-2-sulfatase, and is used to treat Hunter syndrome(Mucopolysaccharoidosis II)).

Imiglucerase (Cerezyme®) is a recombinant analog of humanβ-glucocerebrosidase, and is used to treat Gaucher's disease.

Lapatinib (Tykerb®, Tyverb®) is a dual tyrosine kinase inhibitor thatinterrupts the HER2 growth receptor pathway, and is used to treat breastcancer.

Laronidase (Aldurazyme®) is a recombinant polymorphic variant of thehuman enzyme α-L-iduronidase enzyme, and is used to treatmucopolysaccharoidosis.

Pazopanib (Votrient) is a potent and selective multi-targeted receptortyrosine kinase inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-a/β, andc-kit that blocks tumor growth and inhibits angiogenesis.

Rasburicase (Elitek®) is a recombinant version of urate oxidase, anenzyme which occurs in many mammals but not in humans. It can be used totreat hyperuricemia in patients after cancer treatment.

Reteplase (Retavase®) is a recombinant non-glycosylated form of humantissue plasminogen activator, which has been modified to contain 357 ofthe 527 amino acids of the original protein. It is a thrombolytic drugand used to treat heart attacks.

Rilonacept (Arcalyst®) is a dimeric fusion protein consisting of theextracellular domain of human interleukin-1 receptor and the FC domainof human IgG1 that binds and neutralizes IL-1. It can be used to treatgout.

Sorafenib (Nexavar®) is a small molecular inhibitor of several tyrosineprotein kinases (VEGFR and PDGFR) and Raf kinases. It can be used totreat renal cell carcinoma and liver cancer.

Tenecteplase (TNKase) is a recombinant tissue plasminogen activatoranalog, and is used as a thrombolytic drug.

Vandetanib (Caprelsa®) is an an antagonist of the vascular endothelialgrowth factor receptor (VEGFR) and the epidermal growth factor receptor(EGFR), and can be used to treatment of thyroid cancer.

Vemurafenib (Zelboraf®) is a B-Raf enzyme inhibitor and used fortreatment of melanoma.

Numerous other examples of biologically active molecules will beapparent to the skilled artisan.

Derivatives of a biologicaly active molecule include modifications tothe biologically active molecule to include, for example, a moietyreactive with M or R on the polymer (as described above for compounds(1), (2), and (3))

Preparation of conjugates between a functionalized polymer providedherein and a biologically active molecule or a derivative thereof occursthrough a coupling reaction between the reactive group on the polymerwith a biologically active molecule or derivative thereof. One of skillin the art would appreciate that there are many ways to couple abiologically active molecule or a derivative thereof with the functionalpolymers described herein. For example, a biologically active moleculecan be modified to introduce thiols at the places of reactive aminogroups (e.g., at lysine residues) that can subsequently react with amaleimido or iodoacetamido-group activated functionalized polymerdescribed herein.

A biologically active molecule also can be derivatized to incorporatehydrazo functions that can subsequently react with an aldehyde/ketofunctionalized polymer described herein.

In another method, a biologically active molecule is reacted with areagent that introduces an azido function at amino groups. The resultingazido modified biologically active molecule (e.g., antibody,polypeptide) then can be further reacted with an alkyne-derivatizedfunctionalized polymer to obtain a conjugate via the Click-reaction.

In another method, a biologically active molecule can be reacted with areagent introducing a diene/dienophile that can subsequently react witha diene/dienophile activated functionalized polymer described herein toproduce a conjugate after the Diels-Alder reaction.

In another method, the functionalized polymer is directly linked to abiologically active molecule or a derivative thereof. For example, afunctional polymer (e.g., PEG) containing a carboxyl group that ispreviously activated by a reactive ester group like N-hydroxysuccinimide(NHS) can be conjugated to a biologically active molecule or aderivative thereof.

Generally, a functionalized polymer described herein can include anactivated ester as M-R, and can react randomly with protein amino groups(e.g., lysines) to form covalent linkages. Many activating groups areavailable commercially as a wide variety of leaving groups are known.Non-limiting examples of leaving groups include p-nitrophenol and NHS.Attaching of PEGs to antibodies is often made randomly because the IgGmolecule often demands several polymers to be efficiently protected.

In another method, aldehyde-containing functionalized polymers can forma Schiff s base with amino groups on the protein. The Schiffs base isfurther selectively reduced with sodium cyanoborohydride in a well knownreaction.

In another method, periodate can be used to oxidize carbohydrates on abiologically active molecule to aldehydes, followed by addition ofhydrazo derivatized PEG to be attached. The hydrazo group reacts withthe aldehydes to produce a stable hydrazide link.

In one embodiment, a biologically active molecule conjugate, or apharmaceutically acceptable salt thereof includes a water-soluble,non-peptidic, and non-nucleotidic polymer backbone as in a structure offormula (9):

or a salt thereof,

wherein:

A is the point of covalent bonding to one terminus of the polymerbackbone; E is O or S; K is selected from the group consisting of:alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G isselected from the group consisting of: hydrogen, alkoxy, and ahydrophobic separation handle; Z¹ and Z² are independently selected fromO and NH, wherein only one of Z¹ and Z² can be NH; L is selected fromthe group consisting of: a divalent radical of a nucleoside, alkylene,alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstitutedand substituted arylene; R¹ is absent or a hydrophobic separationhandle, wherein only one of R¹ and G can be a hydrophobic separationhandle; L² is a covalent linking moiety between L on the polymerbackbone and B (e.g., amide, carbamide, ester, oxime, thioether,dithioether, secondary amine, 1,2,4-triazol, or hydrazide linkingmoiety); and B is a biologically active molecule or a derivativethereof.

Such a conjugate can be prepared by reacting a biologically activemolecule or a derivative thereof with a preparation comprising awater-soluble, non-peptidic, and non-nucleotidic polymer backbone havingat least one terminus covalently bonded to a structure of formula (1)(as described above) under conditions suitable for group M to react witha biologically active molecule or the derivative thereof. The group Mcan be hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione,alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone,hydrazide, aminoxy, maleimide, dithiopyridine, or iodoacetamide. In someembodiments, the group reactive with a biologically active molecule orderivative thereof is carboxyl and R is absent or isN-hydroxysuccinimidyl, p-nitrophenyl, or pentachlorophenyl.

In one embodiment, a conjugate, or a pharmaceutically acceptable saltthereof, has a structure of formula (10):

or a salt form thereof,

wherein polymer has a linear, water-soluble, non-peptidic, andnon-nucleotidic polymer backbone (e.g., PEG), wherein each linking groupis bonded at a different terminus of the polymer; E and E¹ areindependently O or S; K and K₁ are independently selected from the groupconsisting of: alkylene, alkyleneoxyalkylene, and oligomericalkyleneoxyalkylene; G and G₁ are independently absent or are selectedfrom the group consisting of: alkoxy and a hydrophobic separationhandle; each pair of Z¹ and Z² and Z³ and Z⁴ are independently selectedfrom 0 and NH, wherein only one of each pair of Z¹ and Z² and Z³ and Z⁴can be NH; L and L¹ are independently selected from the group consistingof: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene,oligomeric alkyleneoxyalkylene, and unsubstituted and substitutedarylene; L² is a covalent linking moiety (e.g., amide, carbamide, ester,oxime, thioether, dithioether, secondary amine, 1,2,4-triazol, orhydrazide linking moiety); between L on the polymer backbone and B; L³is a covalent linking moiety (e.g., amide, carbamide, ester, oxime,thioether, dithioether, secondary amine, 1,2,4-triazol, or hydrazidelinking moiety) between L on the polymer backbone and B¹; and B and B¹are independently a biologically active molecule or a derivative of abiologically active molecule (e.g., a TNF inhibitor, a derivative of aTNF inhibitor, insulin, a derivative of insulin a clotting factor, aderivative of a clotting factor, omalizumab, a derivative of malizumab,a polypeptide that boosts red or white cell production, or a derivativeof a polypeptide that boosts red or white cell product, an antibody, ora derivative of an antibody), a biologic other than the biologicallyactive molecule (e.g., an antibody or a polypeptide other than thebiologically active molecule), a drug, a detectable group, or aseparation moiety, wherein at least one of B and B¹ is a biologicallyactive molecule or a derivative of a biologically active molecule. Forexample, B can be selected from the group consisting of a TNF inhibitor,insulin, omalizumab, a clotting factor, a polypeptide that boosts red orwhite cell production, or an antibody; and B¹ can be a derivative of abiologically active molecule selected from the group group consisting ofa TNF inhibitor, insulin, omalizumab, a clotting factor, a polypeptidethat boosts red or white cell production, and an antibody; B and B¹ canboth be a biologically active molecule; one of B and B¹ is abiologically active molecule and the other is a separation moiety; oneof B and B¹ is a biologically active molecule and the other is adifferent biologic; one of B and B¹ is a biologically active moleculeand the other is a detectable group; or one of B and B¹ is abiologically active molecule and the other is a drug.

Such a conjugate can be prepared by reacting a biologically activemolecule or a derivative thereof with a preparation comprising acompound of formula (2) (as described above) under conditions suitablefor group M to react with a biologically active molecule or thederivative thereof. The group M can be hydroxyl, amine, thiol, carboxyl,aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide,acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide,dithiopyridine, or iodoacetamide. In some embodiments, the groupreactive with a biologically active molecule or derivative thereof iscarboxyl and R is absent or is N-hydroxysuccinimidyl, p-nitrophenyl, orpentachlorophenyl.

In one embodiment, a conjugate, or a pharmaceutically acceptable saltthereof, has a structure of formula (11):

or a salt form thereof,wherein polymer is a linear, water-soluble, non-peptidic, andnon-nucleotidic polymer backbone (e.g., a PEG polymer), wherein M² andthe phosphonate-derived functional group are bonded at a differentterminus of said polymer; E and E¹ are independently O or S; K isselected from the group consisting of: alkylene, alkyleneoxyalkylene,and oligomeric alkyleneoxyalkylene; G is selected from the groupconsisting of: hydrogen, alkoxy, and a hydrophobic separation handle; Z¹and Z² are independently selected from O and NH, wherein only one of Z¹and Z² can be NH; L is selected from the group consisting of: a divalentradical of nucleoside, alkylene, alkyleneoxyalkylene, oligomericalkyleneoxyalkylene, and unsubstituted and substituted arylene; L² is acovalent linking moiety (e.g., amide, carbamide, ester, oxime,thioether, dithioether, secondary amine, 1,2,4-triazol, or hydrazidelinking moiety); between L on the polymer backbone and B; L⁴ is acovalent linking moiety (e.g., amide, carbamide, ester, oxime,thioether, dithioether, secondary amine, 1,2,4-triazol, or hydrazidelinking moiety); between L on the polymer backbone and B¹; and B and B¹are independently a biologically active molecule or a derivative of abiologically active molecule (e.g., a TNF inhibitor, a derivative of aTNF inhibitor, insulin, a derivative of insulin, omalizumab, aderivative of omalizumab, a clotting factor, a derivative of a clottingfactor, a polypeptide that boosts red or white cell production, aderivative of a polypeptide that boosts red or white cell production, anantibody, or a derivative of an antibody), a biologic other than abiologically active molecule, a drug, a detectable group, a separationmoiety, wherein at least one of B and B¹ is a biologically activemolecule or a derivative of a biologically active molecule. For example,B can be a biologically active molecule and B¹ can be a derivative of abiologically active molecule; B and B¹ can both be a biologically activemolecule; one of B and B¹ is a biologically active molecule and theother is a separation moiety; one of B and B¹ is a biologically activemolecule and the other is a different biologic; one of B and B¹ is abiologically active molecule and the other is a detectable group; or oneof B and B1 is a biologically active molecule and the other is a drug.Preparations

Also provided herein are preparations that include a compound orconjugate provided herein. In some embodiments, a preparation caninclude at least 50% of a compound or conjugate by weight. For example,a preparation can include at least 60%, at least 65%, at least 70%, atleast 75% at least 77%, at least 80%, at least 85%, at least 87%, atleast 89%, at least 90%, at least 92%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, and at least 99% by weight of thecompound or conjugate. In some embodiments, the compound or conjugate isessentially pure in the preparation.

A preparation can be a solution, a reaction mixture, a chromatographiceluent, a solid (e.g., a power or crystalline form of the preparation),or any other mixture that includes a compound or conjugate in theappropriate amount or level or purity.

Pharmaceutically Acceptable Salts and Compositions

This document also provides pharmaceutically acceptable salts of thecompounds and conjugates provided herein. Examples of pharmaceuticallyacceptable salts of a compound or a conjugate provided herein includeacid addition salts and base salts of the same.

Suitable acid addition salts are formed from acids which form non-toxicsalts. Examples include, without limitation, the acetate, adipate,aspartate, benzoate, besylate, bicarbonate/carbonate,bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate,esylate, formate, fumarate, gluceptate, gluconate, glucuronate,hexafluorophosphate, hibenzate, hydrochloride/chloride,hydrobromide/bromide, hydroiodide/iodide, hydrogen phosphate,isethionate, D- and L-lactate, malate, maleate, malonate, mesylate,methylsulphate, 2-napsylate, nicotinate, nitrate, orotate, oxalate,palmitate, pamoate, phosphate/hydrogen, phosphate/phosphate dihydrogen,pyroglutamate, saccharate, stearate, succinate, tannate, D- andL-tartrate, 1-hydroxy-2-naphthoate tosylate, and xinafoate salts.

Suitable base salts are formed from bases which form non-toxic salts.Examples include, without limitation, the aluminium, arginine,benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine,magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zincsalts.

Hemisalts of acids and bases may also be formed, for example,hemisulphate and hemicalcium salts.

A conjugate, as provided herein, can be formulated into a pharmaceuticalcomposition that includes an effective amount of a conjugate and apharmaceutically acceptable excipient. Also provided herein arepharmaceutical compositions that include an effective amount of acompound as provided herein, wherein M is a detectable functional group,and a pharmaceutically acceptable excipient.

Non-limiting examples of pharmaceutical excipients suitable foradministration of the conjugates and compounds provided herein includeany such carriers known to those skilled in the art to be suitable forthe particular mode of administration. Pharmaceutically acceptableexcipients include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, self-emulsifying drug delivery systems(SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate,surfactants used in pharmaceutical dosage forms such as Tweens or othersimilar polymeric delivery matrices, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate,sodium-chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethyl cellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, and wool fat.Cyclodextrins such as α-, β, and γ-cyclodextrin, or chemically modifiedderivatives such as hydroxyalkylcyclodextrins, including 2- and3-hydroxypropyl-b-cyclodextrins, or other solubilized derivatives canalso be advantageously used to enhance delivery of a compound orconjugate provided herein. In some embodiments, the excipient is aphysiologically acceptable saline solution.

A pharmaceutical composition can be, in one embodiment, formulated intosuitable pharmaceutical preparations such as solutions, suspensions,tablets, dispersible tablets, pills, capsules, powders, sustainedrelease formulations or elixirs, for oral administration or in sterilesolutions or suspensions for parenteral administration, as well astransdermal ointments, creams, gels, and patch preparations and drypowder inhalers (see, e.g., Ansel Introduction to Pharmaceutical DosageForms, Fourth Edition 1985, 126).

The concentration of a compound or conjugate in a pharmaceuticalcomposition will depend on absorption, inactivation, and excretion ratesof the compound or conjugate, the physicochemical characteristics of thecompound or conjugate, the dosage schedule, and amount administered aswell as other factors known to those of skill in the art.

The pharmaceutical composition may be administered at once, or may bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment is a function of the disease being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular patient, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed compositions.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or conjugates. The pharmaceuticallytherapeutically active compounds or conjugates are, in one embodiment,formulated and administered in unit-dosage forms or multiple-dosageforms. Unit-dose forms as used herein refer to physically discrete unitssuitable for human and animal patients and packaged individually as isknown in the art. Each unit-dose contains a predetermined quantity ofthe therapeutically active compound or conjugate sufficient to producethe desired therapeutic effect, in association with the requiredpharmaceutical carrier, vehicle or diluent. Examples of unit-dose formsinclude ampoules and syringes and individually packaged tablets orcapsules. Unit-dose forms may be administered in fractions or multiplesthereof. A multiple-dose form is a plurality of identical unit-dosageforms packaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit-doses which are not segregated inpackaging.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing a compound orconjugate as provided herein and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, a pharmaceutical composition to be administeredmay also contain minor amounts of nontoxic auxiliary substances such aswetting agents, emulsifying agents, solubilizing agents, pH bufferingagents and the like, for example, acetate, sodium citrate, cyclodextrinederivatives, sorbitan monolaurate, triethanolamine sodium acetate,triethanolamine oleate, and other such agents.

Dosage forms or compositions containing a compound or conjugate providedherein in the range of 0.005% to 100% with the balance made up fromnon-toxic carrier may be prepared. Methods for preparation of thesecompositions are known to those skilled in the art. The contemplatedcompositions may contain 0.001%-100% active ingredient, in oneembodiment 0.1-95%, in another embodiment 75-85%.

Pharmaceutical compositions suitable for the delivery of a compound orconjugate provided herein and methods for their preparation will bereadily apparent to those skilled in the art. Such compositions andmethods for their preparation may be found, for example, in Remington'sPharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

Methods of Treatment

Conjugates of a biologically active molecule or a derivative thereof anda functionalized polymer as provided herein, and combinations thereof(e.g., two or more conjugates of a biologically active molecule or aderivative thereof) can be used to treat a patient as appropriate forthe particular biologically active molecule (e.g., a patient having adisorder selected from the group consisting of an inflammatory disease;diabetes; asthma; hemophilia (e.g., hemophilia A or hemophilia B), adeficiency in a clotting factor (e.g., factor VII deficiency) or otherdisorder affecting coagulation or clotting; with anemia, e.g., a humanpatient having anemia resulting from chemotherapy or kidney disease).

In some embodiments, conjugates of TNF inhibitors or a derivativethereof and a functionalized polymer as provided herein, andcombinations thereof (e.g., two or more conjugates of a TNF inhibitor ora derivative thereof) can be used to treat a patient having aninflammatory disease, e.g., rheumatoid arthritis, psoriasis such asplaque psoriasis or chronic psoriasis, psoriatic arthritis, juvenileidiopathic arthritis, ankylosing spondylitis, Crohn's disease, orulcerative colitis. As used herein, treating inflammatory disease refersto reducing the severity of the disease or slowing progression of thedisease.

The methods described herein include administering to the patient aneffective amount of the conjugate. An effective amount of conjugate(s)or a pharmaceutical formulation containing a conjugate can be any amountthat reduces the severity of the disease or slows progression of thedisease while not inducing significant toxicity in the patient.Effective amounts of conjugates or a pharmaceutical composition can bedetermined by a physician, taking into account various factors that canmodify the action of drugs such as overall health status, body weight,sex, diet, time and route of administration, other medications, and anyother relevant clinical factors.

In some embodiments, conjugates of insulin or a derivative thereof and afunctionalized polymer as provided herein, and combinations thereof(e.g., two or more conjugates of insulin or a derivative thereof) can beused to treat a patient having diabetes, e.g., a human patient havingtype 1 or type 2 diabetes. As used herein, treating diabetes refers tohelping to control blood glucose levels. The methods described hereininclude administering to the patient an effective amount of theconjugate or combinations of conjugates. An effective amount ofconjugate(s) or a pharmaceutical formulation containing a conjugate canbe any amount that helps reduce or maintain blood glucose levels whilenot inducing significant toxicity in the patient. Effective amounts ofconjugates or a pharmaceutical composition can be determined by aphysician, taking into account various factors that can modify theaction of drugs such as overall health status, body weight, sex, diet,time and route of administration, other medications, and any otherrelevant clinical factors.

A conjugate or a pharmaceutical formulation containing a conjugate canbe administered by any route, including, without limitation, oral orparenteral routes of administration such as intravenous, intramuscular,intraperitoneal, subcutaneous, intrathecal, intraarterial, nasal,transdermal (e.g., as a patch), or pulmonary absorption. A conjugatedescribed herein can be formulated as, for example, a solution,suspension, or emulsion with one or more pharmaceutically acceptableexcipients suitable for the particular route of administration.Subcutaneous administration is particularly useful.

In some embodiments, conjugates of omalizumab or a derivative thereofand a functionalized polymer as provided herein, and combinationsthereof (e.g., two or more conjugates of omalizumab or a derivativethereof) can be used to treat a patient having asthma. As used herein,treating asthma refers to reducing the severity of the disease (e.g.,reducing the incidence of asthma exacerbations). Treatment withOmalizumab limits the release of mediators of the allergic response andreduces the number of FIERI receptors on basophils in atopic patients.Treatment with Omalizumab also can increase the expiratory volume in thefirst second of a forced expiration (FEV₁), which is an indicator of theability to exhale rapidly and completely.

The methods described herein include administering to the patient aneffective amount of the conjugate. An effective amount of conjugate(s)or a pharmaceutical formulation containing a conjugate can be any amountthat reduces the severity of asthma while not inducing significanttoxicity in the patient.

In some embodiments, conjugates of a clotting factor or a derivativethereof and a functionalized polymer as provided herein can be used totreat a patient (e.g., a human patient) diagnosed with hemophilia (e.g.,hemophilia A or hemophilia B), a deficiency in a clotting factor (e.g.,factor VII deficiency) or other disorder affecting coagulation orclotting. As used herein, treating refers to reducing the severity ofthe disease or disorder.

Eptacog alfa (activated) (sold by Novo Nordisk) can be used to replacemissing factor VII in patients with factor VII deficiency and also canbe used in hemophilia patients who have developed inhibitors to factorVIII or IX.

Factor VIII deficiency results in hemophilia A and Factor IX deficiencycauses hemophilia B. Moroctocog alfa (glycoprotein (trade name ReFacto®,Xyntha®) are commercially available for Factor VIII replacement therapy.Factor VIII replacement therapy is limited due to development ofhigh-titer inhibitory factor VIII antibodies in some patients. Nonacogalfa (Benefix®) is a human recombinant factor IX product used intreating hemophilia B patients.

Antithrombin is a small glycoprotein that inactivates several enzymes ofcoagulation system. Its activity enhanced by heparin. Antithrombin alfa(e.g., ATryn®) is commercially available.

In some embodiments, conjugates of a polypeptide that boosts red orwhite blood cell production or a derivative thereof and a functionalizedpolymer as provided herein can be used to treat a patient with anemia,e.g., a human patient having anemia resulting from chemotherapy orkidney the group consisting of an inflammatory disease. Conjugates of apolypeptide that boosts white blood cell production or a derivativethereof and a functionalized polymer as provided herein, andcombinations thereof (e.g., two or more conjugates of a polypeptide thatboosts red or white blood cell production or a derivative thereof) canbe used to treat a patient with neutropenia (e.g., neutropenia resultingfrom chemotherapy or bone marrow transplantation) or to increase thenumber of hematopoietic stem cells in the blood before collection. Asused herein, treating anemia or neutropenia refers to reducing theseverity of the disease.

The methods described herein include administering to the patient aneffective amount of the conjugate. An effective amount of a conjugate ora pharmaceutical formulation containing a conjugate can be any amountthat reduces the severity of the disease or disorder while not inducingsignificant toxicity in the patient.

Conjugates of an antibody or a derivative thereof and a functionalizedpolymer as provided herein, and combinations thereof (e.g., two or moreconjugates of an antibody or a derivative thereof) can be used to treata patient, e.g., a human patient. For example, a conjugate that includesAbatacept can be used for the treatment of arthritis (e.g., adultrheumatoid arthritis or JIA). A conjugate that includes Alemtuzumab canbe used for treatment of CLL, CTCL, and T-cell lymphoma, or in aconditioning regimen for bone marrow transplantation, kidneytransplantation, or islet cell transplantation. A conjugate thatincludes Basiliximab can be used to prevent rejection in organtransplantation, (e.g., during kidney transplants). A conjugate thatincludes Belimumab can be used to treat adults with active SLE. Aconjugate that includes Besilesomab can be used to detect inflammatorylesions and metastases. A conjugate that includes Bevacizumab can beused to treat various cancers, including colorectal, lung, breast,glioblastoma, kidney, and ovarian cancer. A conjugate that includesCanakinumab can be used to treat autoinflammatory syndromes includingfamilial cold autoinflammatory syndrome, Muckle-Wells syndrome, andneonatal-onset multisystem inflammatory disease. A conjugate thatincludes Catumaxomab can be used to treat malignant ascites. A conjugatethat includes Cetuximab can be used for treatment of metastaticcolorectal cancer and head and neck cancer. A conjugate that includesDenosumab can be used for the treatment of osteoporosis,treatment-induced bone loss, bone metastases, rheumatoid arthritis,multiple myeloma, and giant cell tumor of bone. A conjugate thatincludes Eculizumab can be used to treat paroxysmal nocturnalhemoglobinuria (PNH). A conjugate that includes Ipilimumab can be usedto treat melanoma (e.g., metastatic melanoma or unresectable melanoma).A conjugate that includes Natalizumab can be used in the treatment ofmultiple sclerosis and Crohn's disease. A conjugate that includesOfatumumab can be used for treating chronic lymphocytic leukemia,follicular non-Hodgkin's lymphoma, diffuse large B cell lymphoma,rheumatoid arthritis, and relapsing remitting multiple sclerosis. Aconjugate that includes Panitumumab can be used to treat metastaticcolorectal cancer. A conjugate that includes Ranibizumab can be used totreat age-related macular degeneration (e.g., AMD and ARMD). A conjugatethat includes Rituximab can be used to treat lymphomas, leukemias,transplant rejection and some autoimmune disorders. A conjugate thatincludes Tocilizumab can be used to treat rheumatoid arthritis (RA) andsystemic juvenile idiopathic arthritis. A conjugate that includesTrastuzumab can be used to treat breast cancer. A conjugate thatincludes Ustekinumab can be used to treat psoriasis. As used herein,treating a particular disease or disorder refers to reducing theseverity of the disease or slowing progression of the disease.

Methods described herein can include monitoring the patient to, forexample, determine if the severity or progression of the disease isimproving with treatment.

Synthesis

Compounds and conjugates provided herein, including salts thereof, canbe prepared using known organic synthesis techniques and can besynthesized according to any of numerous possible synthetic routes. Insome embodiments, a compound or conjugate is prepared using a method asprovided herein.

The reactions for preparing compounds and conjugates provided herein canbe carried out in suitable solvents which can be readily selected by oneof skill in the art of organic synthesis. Suitable solvents can besubstantially non-reactive with the starting materials (reactants), theintermediates, or products at the temperatures at which the reactionsare carried out, e.g., temperatures which can range from the solvent'sfreezing temperature to the solvent's boiling temperature. A givenreaction can be carried out in one solvent or a mixture of more than onesolvent. Depending on the particular reaction step, suitable solventsfor a particular reaction step can be selected by the skilled artisan.

Preparation of compounds can involve the protection and deprotection ofvarious chemical groups. The need for protection and deprotection, andthe selection of appropriate protecting groups, can be readilydetermined by one skilled in the art. The chemistry of protecting groupscan be found, for example, in Protecting Group Chemistry, 1^(st) Ed.,Oxford University Press, 2000; March's Advanced Organic chemistry:Reactions, Mechanisms, and Structure, 5^(th) Ed., Wiley-IntersciencePublication, 2001; and Peturssion, S. et al., “Protecting Groups inCarbohydrate Chemistry,” J. Chem. Educ., 74(11), 1297 (1997) (each ofwhich is incorporated herein by reference in their entirety.

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry, or by chromatographic methods such as high performanceliquid chromatography (HPLC), liquid chromatography-mass spectroscopy(LCMS) or thin layer chromatography (TLC). Compounds can be purified bythose skilled in the art by a variety of methods, including highperformance liquid chromatography (HPLC) (“Preparative LC-MSPurification: Improved Compound Specific Method Optimization” K. F.Blom, et al., J. Combi. Chem. 6(6) (2004), which is incorporated hereinby reference in its entirety) and normal phase silica chromatography.

EXAMPLES Example 1

Synthesis of isopropyl phosphorodichloridite was performed according tothe modified literature procedure of Zwierzak and Koziara, Tetrahedron(1967), 23, 2243-2252.

Dry isopropanol (2 mole) in 100 mL of dry diethyl ether was addeddropwise to the vigorously stirred mixture of phosphorus trichloride (4mole) in 200 mL of diethyl ether at −20° C. The mixture was allowed towarm up to room temperature, and was stirred at this temperature for 4hours. The mixture was fractionally distilled at normal pressure toobtain the title product by collecting a colorless fraction having bp.120-125° C.

Example 2

Synthesis of Isopropyl, N,N-Diisopropylphosphoramidochloridite.

Isopropyl phosphorodichloridite (56.5 g, 0.5 mole) in dry diethyl ether(300 mL) was placed in a 1 L round bottom flask and cooled to −30° C. Tothis vigorously stirred solution, dry diisopropyl amine (109 g, 1 mole)in diethyl ether (200 mL) was added dropwise (2 hr) at the above lowtemperature. The mixture was then allowed to warm to room temperatureand was left at this temperature overnight. The thick white cake ofdiisopropylammonium hydrochloride was filtered off on a large filterfunnel and washed with two portions of ether. The combined ether phasewas evaporated and the residue was distilled at lowered pressurecollecting a fraction boiling at 83-85° C. (15 Torr).

Example 3

The following is a general procedure used to prepare differentphosphoramidite reagents.

An appropriately protected alcohol (1 eq.) was placed in a round bottomflask and dried by coevaporation with toluene. The residue was dissolvedin dry dichloromethane (DCM) (5 mL/mmole), and dry triethylamine (TEA)(4 eq.) was added in one portion. To this solution, stirred at roomtemperature, isopropyl, N,N-diisopropylphosphoramidochloridite (1.5 eq.)in DCM (2 mL/mmole) was added dropwise. The mixture was stirred anadditional 30 minutes and when TLC (DCM:Methanol:TEA 95:4:1) showed acomplete consumption of the starting alcohol, methanol (5 eq.) was addedin one portion and the mixture was stirred further for 30 minutes. Thereaction mixture was partitioned between dichloromethane and an aqueoussolution of sodium bicarbonate. The organic extracts were combined,evaporated, dried by coevaporation with toluene, and purified by flashcolumn chromatography on silica gel using a gradient of ethyl acetate inhexane with addition of 2% TEA as an eluent. The appropriate fractionswere collected, and evaporated. The residual solvents were removed underhigh vacuum, yielding products in the form of thick oils.

The following starting alcohols were prepared according to the publishedliterature and converted to the respective isopropyl phosphoramiditesusing the procedure outlined above:

1) An amidite useful for introduction of an amino group was prepared bymodification of the method of Gaur, Nucleosides, Nucleotides & NucleicAcids (1991), 10, (4), 895-909:

2) An amidite useful for introduction of an aminoxy group was preparedin a process similar to the synthesis of described above for aminatingan amidite. The starting aminoxy alcohol was prepared from 6-bromhexanolaccording to Khomutov, Zhurnal Obshch Khimyi 1961, 31, 1992-1995:

3) An amidite useful for introduction of a hydrazo group was preparedusing a modified method of Raddetz et al., Nucleic Acids Res. 30, (21),4793-4802:

4) An amidite useful for introduction of a thiol was obtained followingthe procedure described by Connolly and Rider in Nucleic Acids Res.1985, 13, (12), 4485-4502:

5) An amidite useful for introduction of a carboxyl group was preparedutilizing a chlorotrityl group for protection of a carboxyl group as wasdescribed by Kachalova et al. Helv. Chim. Acta 2002, 85, 2409-2416:

6) An amidite useful for introduction of biotin was prepared followingthe methodology described by Krempsky et al., Tet. Lett., (1996), 37,(12), 4313-4316:

7) The following amidite, prepared in a method analogous to onedescribed by Singh et al. J. Org. Chem. 2004, 69, 8544-8546, was usedfor incorporation of an aliphatic aldehyde group. The aldehyde (as anamide of glyoxalic acid) was obtained after oxidative cleavage of thecis-amine alcohol group:

8) The following amidite can be used for incorporation of an aliphaticaldehyde group. This reagent was made in analogy to Spinelli et al.Nucleosides, Nucleotides and Nucleic Acids 2007, 26, 883-887. Thealdehyde was obtained after acidic hydrolysis of the acetal bond andoxidation of the cis diol system:

9) The following amidite, prepared by modification of method ofPodyminogin et al., Nucl. Acids Res. 2001, 29, (24), 5090-5098, can beused for incorporation of an aromatic aldehyde group:

10) An amidite allowing for introduction of an active ester in a singlechemical step was prepared analogously to the method described in U.S.Pat. No. 6,320,041:

Example 4

The following is a general procedure that was used for the synthesis ofpure monosubstituted PEG polymers.

This method utilizes non-derivatized PEG used in excess over theselected phosphoramidite reagent in order to obtain better selectivity.Amidites containing a hydrophobic moiety, like trityl, substitutedtrityl, long chain fatty esters, or acetals introduce a separationhandle that can be used for reversed-phase based chromatographicseparation of the product.

Thus PEG 6000 (60 g, 10 mmole, 2 eq.) was dried by double coevaporationwith toluene (heating was applied in order to dissolve all material).The residue was dissolved in dry acetonitrile (50 mL) and an appropriatephosphoramidite (5 mmole, 1 eq.) in dry acetonitrile (30 mL) was addedin one portion. To this clear solution, stirred at room temperature, asolution of 4,5-dicyanoimidazole (5.9 g, 50 mmol, 10 eq.) inacetonitrile was added in a single portion and the mixture was stirredfor 15 minutes. Reaction was stopped by addition of a solution of iodine(0.1 M, 1.2 eq.) in THF:pyridine:water (10:10:1) and the brownishsolution was stirred for 3 minutes. Aqueous solution of sodium bisulfitewas added in small portions until decolorization of the reactionmixture. After evaporation of most of the volatile matter, the residuewas dissolved in dichloromethane and extracted with a saturated aqueoussolution of sodium bicarbonate. The evaporated organic phase was driedby coevaporation with toluene, and the residue was crystallized fromisopropanol. The isolated crystals were dissolved in a small amount ofdichloromethane and precipitated by addition of diethyl ether. The finalmixture, being a composition containing a free non-derivatized PEG,mono- and bis-derivatized PEG, was preperatively purified byreverse-phase chromatography. Fractions containing the desired productwere evaporated, dried and precipitated from diethyl ether. When removalof the acid labile separation handle was required the polymer isdissolved in isopropanol (with slight heating) and a 5% solution oftrichloracetic acid in isopropanol was added. The mixture is cooled downin the freezer to obtain a deprotected crystalline product. If a TLCtest (silicagel plates in 10% MeOH:DCM) for the purity of the productstill showed the presence of the trityl protecting group, the procedureof acidic deprotection was repeated. Deprotection of otherfunctionalities were performed following the literature procedures asfar as was possible, but with some modifications:

1) Trityl protected thiol modified polymer was deprotected with a silvernitrate solution in water. After 30 minutes the solution was treatedwith dithiotreithiol (2 eq. to the amount of silver ions added) and thepH was raised to 9.0 by addition of potassium carbonate. After 30minutes, the mixture was filtered through a pad of celite and extractedby dichloromethane. The organic phase was evaporated and thiolated PEGwas crystallized from isopropanol.

2) Polymers modified with different aldehyde-introducingphosphoramidites were first treated with acid to remove the trityl orthe acetal function. In the case of reagent 7 (Example 3) the FMOC groupwas removed by treatment of the polymer with 10% piperidine in DCM for 4hours, followed by quick evaporation of the volatile matter andcrystallization of the residue from isopropanol. The final conversion ofthe cis-amino alcohol or cis-diol to the aldehyde was done by means of10 mM sodium periodate.

Example 5

Pure, mono-substituted PEG molecules used for further derivatizationwere prepared as follows.

Certain PEG derivatives can be prepared in a large scale and atreasonably low cost. These derivatives can be regarded by themselves asvaluable modifications, but also as a good monovalent starting material,well suited for further derivatization. This Example provides for thepreparation of three such derivatives:

1) Mono DMTr-O-Substituted, Linear PEG.

An appropriate PEG (100 g, 2 eq.) was dried by double coevaporation withdry pyridine, dissolved in pyridine (100 mL), and DMTrCl (1 eq.) wasadded to the stirred mixture at room temperature. The yellow solutionwas stirred for 24 hours and methanol (10 mL) was added. The reactionmixture was stirred for an additional 60 minutes, evaporated, dissolvedin dichloromethane, and treated with an aqueous solution of sodiumbicarbonate. The organic phase was evaporated, dried by coevaporationwith toluene, and all PEG was isolated after precipitation from diethylether. The collected mixture of PEG's was purified by reverse-phasechromatography.

2) Mono MMTr-NH-Substituted, Linear PEG.

A commercial monoamino substituted PEG (1 eq.) or a material preparedaccording to any of existing procedures, was dried by doublecoevaporation with dry pyridine. The residue was dissolved in pyridine(10 mL/mmole of PEG) and trimethylchlorosilane (TMSCl) (4 eq.) wasadded. The mixture was stirred at room temperature for 4 hours andMMTrCl (1.5 eq.) was added. The reaction mixture was stirred overnight,and then methanol (50 eq.) was added and the mixture was stirred for anadditional two hours. The total PEG was isolated after evaporation,drying by coevaporation with toluene, crystallization from isopropanoland precipitation from diethylether. The collected mixture of PEG's waspurified by reverse-phase chromatography.

3) Mono Tr-S-Substituted, Linear PEG.

An appropriate, non-derivatized PEG was dried by coevaporation withtoluene and dissolved in dry DMF (5 mL/mmole). To this stirred solutionat room temperature a preformed solution of phosphoroxychloride (0.4eq.) in dry DMF (5 mL/mmole) was added and stirring was continued for 6hours. Most of the solvent was then evaporated, and the residue wastreated using a saturated aqueous sodium bicarbonate solution.Polyethylene glycol was extracted with dichloromethane and concentratedby evaporation of all volatile matter. The reaction mixture, containingthe monochloro-derivatized PEG was dissolved in ethanol (10 mL/mmole).Triphenylmethyl mercaptan (1.3 eq. to the starting PEG) was suspended inethanol (10 mL/mmole) and converted to the sodium salt by addition of anequivalent amount of sodium hydroxide dissolved in a small amount ofwater. The salt was combined with the ethanolic solution of PEG and themixture was stirred overnight. After evaporation of ethanol, the residuewas partitioned between dichloromethane and aqueous sodium bicarbonate.The organic phase was evaporated and the residue was crystallized fromisopropanol, followed by precipitation of total PEG from diethyl ether.The collected mixture of PEG's was purified by reverse-phasechromatography.

Example 6

a) Conversion of monosubstituted PEG to bis-derivatized PEG wasperformed using the following method.

A one equivalent of commercial methyl PEG (mPEG), (or any of partiallyprotected PEG's from the Examples 4 or 5), was dried by coevaporationwith toluene, and the polymer was dissolved in dry acetonitrile (5mL/mmole). The modifying phosphoramidite (2-3 eq.) in dry acetonitrile(3 mL/mmole) was added, followed by a suitable activator (10-15 eq.) andthe reaction mixture was stirred at RT for 15 minutes. At this point allreactions were analyzed by fast reversed-phase analytical chromatographythat showed disappearance of all starting material and formation of morehydrophobic product. Reaction was quenched by addition of an oxidizingiodine solution (1.5 eq. to the amount of the phosphoramidite) ort-butylhydrogenperoxide (4 eq. to the amount of the phosphoramidite) incases when the synthesized product did not tolerate iodine or water. Theiodine-treated reaction mixtures were evaporated, dissolved indichloromethane, decolorized with bisulfite, and the organic phase wasevaporated. The t-butylhydrogenperoxide treated mixtures were evaporateddirectly. Both mixtures were partially purified by crystallization fromisopropanol, followed by precipitation from diethyl ether to obtain apure, mono-functionalized, bis-substituted PEG.

b) An azeotropically dried PEG was dissolved in dry acetonitrile (5mL/mmole). The modifying phosphoramidite (1.0 eq.) in dry acetonitrile(3 mL/mmole) was added, followed by a suitable activator (10-15 eq.) andthe reaction mixture was stirred at RT for 30 minutes. Reaction wasquenched by addition of an oxidizing iodine solution (1.5 eq. to theamount of the phosphoramidite) or t-butyl hydrogenperoxide (4 eq. to theamount of the phosphoramidite) in cases when the synthesized product didnot tolerate iodine or water. The iodine-treated reaction mixtures wereevaporated, dissolved in dichloromethane, decolorized with bisulfite,and the organic phase was evaporated. The t-butylhydrogenperoxidetreated mixtures were evaporated directly. Both mixtures were partiallypurified by crystallization from isopropanol, followed by precipitationfrom diethyl ether to obtain reaction mixture free of low molecularcomponents. This mixture was separated on an analytical and on apreparative RP chromatographic system which allow for isolation of puremonosubstituted product. This methodology could be easy applied foramidites introducing amino, hydroxylamino, hydrazo, carboxyl, aldehydeand the biotin group. The isolated products could be deprotected upontreatment with appropriate acidic conditions, followed by precipitationof the deprotected PEG from diethylether, or the non-deprotectedmaterial could be used in preparation of hetero-bis-functionalizedpolymer as presented below.

c) Using the above idea of conversion of PEG diol into a pure,mono-functionalized product from Example 6 b) with subsequentderivatization with another reagent, the following compounds wereprepared.

1) MMTr-NH—(CH₂)₅—O—PO—(O-iPr)O-PEG-O(O-iPr)—PO—O—(CH₂)₆—O—NH-MMTr

2)MMTr-NH—(CH₂)₅—O—PO—(O-iPr)O-PEG-O(O-iPr)—PO—O—CH(CH₃)(CH₂)₂—COO—ClTr.

The second derivatization were performed as in Example 6a) using excessof the second reagent over PEG to insure quantitative conversion to thedouble functionalized product The final compound was analyzed by HPLC,but any preparative chromatography at this stage was not needed.

Example 7

The following describes methods used to introduce functional groupswhich were not stable in the presence of the phosphoramidite group.

Diethylene glycol protected on one end with a DMTr group, and containinga H-phosphonate group at the other end, was synthesized from atritylated diol by a standard PCl₃/triazole method according to Garegget al., Chem. Scr. 1986, 26, 59-62.

The above H-phosphonate (3 eq.) and mPEG (1 eq.) were dried by repeatedcoevaporation with dry pyridine. The residue was dissolved in pyridine(10 mL/mmole) and treated with pivaloyl chloride (9 eq.). The mixturewas stirred at room temperature for 2 hours and the reaction wasquenched by addition of triethylammonium bicarbonate (TEAB) (1 M, 5mL/mmole). The reaction mixture was concentrated and partitioned betweendichloromethane and diluted TEAB. The organic phase was evaporated, theresidue was dried by repeated coevaporation with toluene, and total PEGwas purified by precipitation from diethyl ether. The isolatedprecipitate was filtered, dissolved in pyridine/carbon tetrachloride 2:1and 1-amino-6-azidohexane (4 eq.) was added. The stirred mixture wasleft overnight at room temperature. The mixture was evaporated,coevaporated with water, and the residual material was treated with anaqueous ammonia solution (25%, 20 mL/mmole) for 4 hours at roomtemperature in order to cleave the residual non-oxidized H-phosphonatedimer. Ammonia was evaporated and the resulting crude product waspurified by means of preparative reverse-phase chromatography. Finalremoval of the DMTr group and precipitation of the PEG gave the pureazido modified product.

Example 8. Conjugation of Omalizumab

The following describes methods used to conjugate omalizumab with amodified PEG 20K polymer.

Selective DMTr Protection of One End of the PEG Chain.

Commercial PEG 20,000 (20K) (20 g, 1 mmol) was dried by repeatedco-evaporation with toluene (3×200 mL) followed by co-evaporation withdichlormethane (200 mL). The residue was dissolved in DCM anddimethylaminopyridine DMAP (0.2 mmol), dry triethylamine (5 mm ol) andDMTrCl (1 mmol) were added. The mixture was stirred overnight at roomtemperature and all volatile matters were evaporated under reducedpressure. The solidified products were dissolved in a minimal volume ofacetonitrile (25 mL) and cold (−20 deg C.) isopropanol (200 mL) wasgradually poured into this solution with magnetic stirring. Thecrystallized mixture of PEG derivatives was vacuum filtered, washed withcold isopropanol, and dried overnight at reduced pressure.

A 3 gram portion of this material was dissolved in 10 mL of water/EtOH(4:1) and applied on a manually packed Hamilton PRP-1 Polystyrene column(3×10 cm), washed with ethanol, and equilibrated with 20% EtOH/watercontaining 0.2% conc ammonia solution.

In order to ensure that all non-derivatized PEG has been eluted out fromthe column, elution of all products was done using a gradient of ethanolin water: Solvent A: 20% EtOH+0.2% ammonia; solvent B: 100% EtOH+0.2%ammonia. The run was done very slowly and the starting, non-derivatizedPEG came out at a solvent mixture of 22-25% of B, while the broad peakof mono tritylated product came within a solvent mixture of 46-55% of B.It should be noted that only the first part of this broad peak: at asolvent mixture of 46-50% of B was used in further synthetic steps. Itimplies, most probably, that reverse phase (RP) separation of tritylatedPEG fractionates the PEG molecules according to their molecular weight,with larger molecules coming first. All runs were performed at the flowof 2 mL/min.

The collected fractions were examined for the presence of PEG using acolorimetric test using a barium chloride and KI/iodine solution. Thistest together with the chromatogram showing the absorbance of the DMTrgroup indicated that the isolated product was the mono-DMTr protectedPEG. This material was free of bis-tritylated product and free ofnon-derivatized PEG. The purity of the isolated material was confirmedby a separate HPLC analysis on a C-18 RP column. The product wasconcentrated to dryness, dissolved in a minimal amount of acetonitrile(1 mL), precipitated from diethyl ether (50 mL) and dried under vacuum.

Synthesis of the Hetero Bi-Functional Active Ester Form of PEG

Mono-DMTr protected PEG (1.0 g, 0.05 mmol) was dried by doublecoevaporation with dry acetonitrile (2×30 mL) and previously synthesizedamidite (See Example 3) that introduces NHS-ester function (0.25 mmol,125 mg) was added followed by addition of tetrazol solution (0.1 M) indry acetonitrile (13 mL, 1.3 mmol, 5 eq per amidite).

The mixture was stirred at RT for 30 min and an oxidizing agent t-butylhydrogen peroxide solution in dry toluene (1M, 2 mL, 8 eq) was added ina single portion. Stirring was continued for an additional 30 minutesand the volatile components were evaporated at reduced pressure. Theresidue was dissolved in 2 mL of acetonitrile and the non-solubilizedtetrazole was spun down. The clear upper solution was transferred to alarger 50 mL Falcon tube and cold isopropanol (−20° C.) was graduallyadded causing crystallization of the PEG molecule. This material wasspun down, washed again with cold isopropanol, and dried under vacuum(oil pump). The product was obtained in the form of white fluffycrystals.

Conjugation of Omalizumab to PEG Reagent.

Conjugation of PEG to IgG omalizumab was performed following themethodology described in J. Decruex, R. Vanbever and, P. R. Crocker,Bioconjugate Chem. (2008) 19: 2088-2094.

Briefly, omalizumab (300 mg; SEQ ID NO:1) was dissolved in water (5 ml)and portionized into separate Sörsted tubes containing 15 mg of IgGeach. One of the tubes was diluted with (PBS) phosphate buffer (0.2 M,pH 7.3) to 1 mL and placed on a NAP 10 desalting column previouslyequilibrated with the same buffer. The desalted protein was isolatedwith 1.5 mL of the buffer. To a portion of the desalted IgG (0.5 ml, 5mg, 0.033 micromol), PEG reagent (20 mg, ca 5 micromol, 30 eq) was addedand the mixture was left for 180 min with occasional shaking After thistime a solution of glycine (0.1 M) in phosphate buffer (0.2 mL) wasadded to quench any non-reacted PEG reagent.

The conjugate was analyzed by the following methods:

1) SDS electrophoresis 7.5% acrylamide gel for 90 min and 180 V.Proteins were stained with Coomassie blue (see FIG. 2).

2) The above gel was additionally stained by using a solution of bariumchloride and iodine which is known to selectively label PEG containingmolecules in a brown to black color dependent on the amount of PEG (thisis the main method for detection of non-derivatized PEG). During thisstep the previously Coomassie stained proteins did not change theircolor (see FIG. 3).

3) SDS gel electrophoresis was also run using lower amounts of bothreference IgG and conjugation reaction, and was performed for a longertime (180 min) and higher voltage (220 V) in order to achieve betterconjugate separation. The gel was stained using Coomassie blue. FIG. 4shows the conjugate on the left side and free IgG on the right.

4) Using the methodology described above, two additional conjugationreactions were also performed. Both reactions were made starting from 5mg of omalizumab in PBS buffer. For 10 equivalents excess of PEGreagent, a portion of 7.5 mg of PEG NHS was added, and for a 5equivalents reaction, 3.75 mg PEG was added. Both reactions werequenched after 3 hours and analyzed together alongside the previous 30equivalents reaction by SDS gel electrophoresis using 8% acrylamide.This time runs times were 180 min to achieve better separation.

FIG. 5 shows the results of the reactions as follows:

Lane 1—2 μg of IgG conjugate obtained by using 5 eq of PEG reagent

Lane 2—2 μg of IgG conjugate obtained by using 10 eq of PEG reagent

Lane 3—2 μg of IgG conjugate obtained by using 30 eq of PEG reagent

Lane 4—2 μg of IgG starting material

Lane 5—protein mass standards

Lane 6—5 μg of IgG conjugate obtained by using 5 eq of PEG reagent

Lane 7—5 μg of IgG conjugate obtained by using 10 eq of PEG reagent

Lane 8—5 μg of IgG conjugate obtained by using 30 eq of PEG reagent

Lane 9—5 μg of IgG starting material

Lane 10—protein mass standards

5) Analysis of the reaction mixture was also performed using gelpermeation chromatography. This analysis was performed using a ZorbaxGF-450 HPLC column and phosphate buffer (0.2 M, pH 7.0) as eluent usinga 1.0 mL/min flow. FIG. 6 shows that the pool of omalizumab conjugateswas well separated from the starting non-conjugated omalizumab.

Example 9. Pegylation of Lysozyme and Insulin

5 mg of each protein (0.35 μmol of lysozyme (Mw=14,300) and 0.86 μmol ofinsulin (Mw=5,808; SEQ ID NO. 2)) were dissolved in phosphate buffer(0.2 M, pH 7.4) and PEGylating (in form of previously described NHSreagent—see Example 8) 20 kD was added in 5-fold excess. The reactionwas kept overnight and was quenched by addition of excess glycine. Asample of both starting protein and pegylated reaction mixtures weretested on a Zorbax GF 450 gel filtration HPLC column, with 280 nmdetection, to determine the degree of derivatization.

As shown in FIG. 8, the normalized chromatogram shows lysozyme (dashedline) and lysozyme pegylation reaction (solid line). The double peak (atapproximately 10-12 min) represents multiply pegylated lysozyme, thepeak at 13 min is the residual PEG reagent, and the last peak representsunreacted protein.

As shown in FIG. 9, the chromatogram shows insulin (dashed line) andinsulin pegylation reaction mixture (solid line). For an unknown reason,insulin (despite its formally lower Mw) has a shorter retention timethan lysosome, and it co-elutes with the residual PEG reagent. Thefastest running multiple peak (10-12 min) represents pegylated insulin.

Example 10. Pegylation of Etanercept (ENBREL®)

Etanercept (SEQ ID NO:3) was pegylated in the same manner as describedin Example 8. Both omalizumab and etancercept have very similar Mw, butetanercept is not a monoclonal Ab, but is instead a soluble receptor.The different shapes of these two proteins is probably the reason why ingel filtration HPLC analysis free etanercept is running closer to itspegylated conjugate than in the case of omalizumab.

SDS electrophoresis 7.5% acrylamide gel run for 90 min and at 180 V.Proteins were stained with Coomassie blue. FIG. 11 shows the results ofthe reactions as follows:

-   -   1) Lane 8 shows pegylation of etancercept using 5 eq of PEG        reagent—the residual amount of free etancercept also appears in        this lane.    -   2) Lane 5 shows pegylation using 10 eq of PEG reagent—free        etancercept is not visible any longer—at least three different        bands of pegylated etancercept are shown.    -   3) Lane 1 shows pegylation with 30 eq of PEG reagent. All        etancercept is pegylated.    -   4) Lane 4 is a commercial protein ladder.

FIG. 12 shows the normalized chromatogram showing free etanercept(dashed line), and pegylated etanercept after reaction with 10equivalents of the pegylating reagent (solid line). During thepreparative HPLC run, half minute long fractions were collected startingfrom 7.5 min, and ending at 10.0 minutes. The content of proteins wasmeasured photometrically at 280 nm. To avoid all possible contaminationof free etanercept in the conjugate fraction, a fraction from 8.0 min to8.5 min was chosen for the further studies.

Example 11. Evaluation of Pegylated Etanercept (ENBREL®) by ELISA

Material and Methods

ENBREL® (etanercept; Wyeth Europa Ltd, Berkshire, UK) was conjugated toa 20 KDa PEG (PEG20) as described in Example 8. The 20 KDa PEG wasconjugated at 5 times (5 eq) or 10 times (10 eq) molar excess comparedto etanercept.

Recombinant human TNF-alpha (rhTNF-alpha; AbCam PLC, Cambridge, UK; at afixed concentration of 2.9 ng/ml) was mixed with differentconcentrations (29000 ng/ml-56.6 pg/ml) of non-PEGylated etanercept,PEG20-etanercept (5 eq) or PEG20-etanercept (10 eq). The pre-incubationmixtures were incubated in a refrigerator at 4-8° C. overnight. Theamount of unbound rhTNF-alpha was analysed with a sensitiveenzyme-linked immunosorbent assay (ELISA). In brief, 96-well plates(MaxiSorb, Nunc, Roskilde, Denmark) were used for the ELISA. Plates werecoated with two anti-TNF-alpha monoclonal antibodies (TNF3/4, MabtechAB, Nacka Strand, Sweden) diluted in PBS, pH 7.4, to a finalconcentration of 2 μg/ml. The 96-well plates were incubated in arefrigerator at 4-8° C. overnight. The 96-well plates were thereafterrepeatedly washed with PBS/0.05% Tween 20. rhTNF-alpha standard(dilution series 1:1) and the pre-incubation mixture containingrhTNF-alpha/PEG20-etanercept (5 or 10 eq) or rhTNF-alpha/etanercept(non-PEGylated) diluted in PBS/0.1% BSA were thereafter added to theplates and incubated for 2 hours at room temperature. The plates wererepeatedly washed with PBS/0.05% Tween 20 and thereafter incubated 1hour at room temperature with 1 μg/ml of a biotin-conjugatedanti-TNF-alpha antibody (TNF5, Mabtech AB, Nacka Strand, Sweden) dilutedin PBS/0.1% BSA/0.05% Tween 20. Finally, streptavidine-HRP (Mabtech AB,Nacka Strand, Sweden) diluted 1:1000 in PBS/0.1% BSA/0.05% Tween 20 wasadded to the plates and incubated 1 hour following a repeated wash stepwith PBS/0.05% Tween. A developer (3, 3′,5,5′ tetramethylbenzidine, TMB;Sigma-Aldrich, St Louis, Mich., USA) was added after a final repeatedwash with PBS/0.05% Tween 20. A blue color reaction developed and thereaction was terminated by the addition of 1 M H₂SO₄ 15-20 minutes. Theoptical density of the resultant yellow color was measured in aspectrophotometer at 450 nm.

The resulting ODs were plotted in GraphPad Prism 6.0 and fitted to a4-parameter logistic curve fitting algorithm.

Results

The covalent attachment of PEG20 did not disturb etanercepts bindingcapacity of rhTNF-alpha. IC₅₀ values of etanercept (with or withoutPEG20) dilution curves were calculated and are shown in FIG. 13. Theresults demonstrate that the IC₅₀ values of PEG20-etanercept (5 or 10eq) are less than 2-fold higher compared to non-PEGylated etanercept(FIG. 13).

The IC₅₀ concentrations of non-PEGylated and PEGylated etanerceptclearly indicate that a 20 KDa PEG does not significantly impair thebinding and neutralization of TNF-α by etanercept. In addition, the useof 5 or 10 equivalents of 20 KDa PEG during the conjugation process toetanercept does not deteriorate the inhibition of TNF-α.

Example 12. Biosensor Evaluation of the Binding of Pegylated Omalizumabto Human IgE

The method was based on LigandTracer technology developed by RidgeViewInstruments AB, Uppsala, Sweden (www.ridgeview.eu).

The experiment was performed on rotated petri dishes. 2 μg of human IgE(AbCam, Cambridge, UK) was adsorbed on the plastic surface and allowedto dry over-night. 100 μg omalizumab (Genentech, CA, USA) was labeledwith ¹²⁵I (13.1 MBq/100 μg) according to the chloramine T method.¹²⁵I-labeled omalizumab (10 nM) was then added to the IgE on therotating petri dish. After approximately 3½ hour the labeled antibodywas exchanged with a buffer (see FIG. 14). Unlabeled omalizumab orPEG20-omalizumab (approx. 200 nM; see Example 8) was added to evaluatethe ability to compete and displace ¹²⁵I-labeled omalizumab bound toIgE. The upper curve (blue) shows the binding of ¹²⁵I-labeled omalizumabto IgE without competition (FIG. 14). The lower curve (red) shows how¹²⁵I-labeled omalizumab bound to IgE is displaced and competed out byunlabeled omalizumab. The curve in between (black) demonstrates thatPEG20-omalizumab (30 eq) has the ability to displace and partiallycompete out ¹²⁵I-labeled omalizumab bound to IgE.

Example 13. Synthesis of a Reagent for Introduction of an Aminoxy Group,Containing a Linker Unit Similar to the Polyethylene Glycol Chain

Monotosylate of Diethylene Glycol.

To an ice chilled solution of diethyleneglycol (19 mL, 200 mmol) inanhydrous pyridine (8.1 mL, 100 mmol) and dichlormethane (50 ml), wasadded in a single portion of a solution of p-toluenesulfonyl chloride(7.6 g, 40 mmol) in dichlormethane (30 mL). The mixture was stirred atroom temperature overnight and partitioned between a saturated solutionof sodium bicarbonate and dichloromethane (2×200 mL). The combinedorganic phases were evaporated and coevaporated with toluene (2×100 mL)to remove pyridine and traces of water. The remaining yellow oil waspurified using flash silica gel chromatography, yielding the titleproduct in the form of colorless oil in a 67% yield.

N-2-(2-Hydroxyethoxy)Ethoxyphtalimide.

To a stirred solution of N-hydroxyphtalimide (4.3 g, 26.2 mmol) in dryDMF (mL) was added DBU (4.0 g, 26.2 mmol). To this dark red solution wasadded monotosylate of diethylene glycol (6.8 g, 26.2 mmol). The reactionmixture was heated at 80° C. overnight, concentrated in vacuo, dissolvedin ethyl acetate (200 mL) and extracted with saturated sodiumbicarbonate until the aqueous phase was colorless. The organic phase wasevaporated and dried by coevaporation with toluene. The title productwas obtained after purification by flash silica gel chromatography aswhite solid. Yield 74%.

Hydrazinolysis of N-2-(2-Hydroxyethoxy)Ethoxyphtalimide.

To a stirred solution of N-2-(2-Hydroxyethoxy)ethoxyphtalimide (4.85 g,19.3 mmol) in ethanol was added hydrazine hydrate (25 mmol) and themixture was stirred at room temperature for 1 hr. The white solid formedduring this reaction was filtered and the filtrate was evaporated todryness and coevaporated with toluene (3×50 mL) yielding an oil, whichwas used further without purification.

Tritylation of Alkoxy Amine.

The oily compound obtained above was dissolved in dry pyridine (100 mL)and to this stirred solution trimethylchlorosilane (4.9 mL, 38.6 mmol)was added in one portion. The mixture was stirred for 30 min andmonomethoxy trityl chloride (MMTrCl) (8.9 g, 29 mmol) was added. Thismixture was stirred at room temperature overnight and was desilylated byaddition of methanol (50 mL). The clear mixture was stirred for 2 hr.,concentrated at reduced pressure and the residue was partitioned betweensaturated sodium bicarbonate and chloroform. The combined chloroformextracts (2×200 mL) were evaporated, coevaporated with toluene (2×100mL), and the residual oil was purified by flush silica gelchromatography, yielding the product in form of oil. Yield 77%.

Synthesis of the Title Phosphoramidite.

The N-MMTr protected aminoxy alcohol (5.55 g, 14.8 mmol) was dried bycoevaporation with toluene (2×50 mL), dissolved in dry dichloromethane(100 mL) and dry triethylamine (6.0 mL, 59.2 mmol) was added followed byisopropyl, N,N-diisopropylphosphoramidochloridite (22.2 mmol). The restof this reaction was made in accordance with the general description ofphosphitylation process as in Example 3. The final product was obtainedin 81% yield in the form of a slightly yellow oil.

Example 14

This example provides a process for preparing a reagent which introducesan aliphatic aldehyde group to a previously monofunctionalizedpolyethylene glycol (e.g., a DMTr containing polymer). The fullyderivatized PEG is deprotected in a single step upon treatment withdiluted hydrochloric acid.

2-(2,2-Dimethoxy-Ethoxy)-Ethanol.

This compound was obtained from ethylene glycol and2-chloro-1,1-dimethoxy-ethane following procedure described by Lei Taoet al. J. Am. Chem. Soc. 2004, 126, 13220-13221. The final product wastaken to the following reaction without purification.

The hydroxyl acetal was converted to the title product following themethodology from Example 3, yielding, upon silica gel purification theproduct in a 61% yield as colorless oil.

-   1) An essentially pure, modified polymer, comprising a water    soluble, non peptidic and non nucleotidic, linear or branched    polymer backbone, containing from 2 and up to 100 termini, and    having at least one terminus being covalently bonded to the    structure:

wherein:

-   -   A is the point of bonding to the terminus of the polymer        backbone;    -   E is oxygen or sulfur;        -   K is selected from the group consisting of linear alkyl,            branched alkyl, alkyloxyalkyl, or oligomeric form of            alkyloxyalkyl;        -   G is none, or is selected from the group consisting of an            alkoxy, trityloxy or substituted trityloxy group;        -   Z₁ and Z₂ are O or NH in such a way that both Z₁ and Z₂ may            be O, but when Z₁ is NH then Z₂ is O, and when Z₂ is NH then            Z₁ is O.        -   L is selected from the group consisting of linear alkyl,            branched alkyl, nucleoside, alkyloxyalkyl, oligomeric form            of alkyloxyalkyl, aryl and substituted aryl;        -   M is a group reactive with a biologically active molecule or            detectable functional group;        -   R is selected from a group consisting of protecting groups,            hydrophobic separation handles, activating groups, hydrogen            or none, R₁ is a hydrophobic separation handle or none, and            both R and R₁ can coexist providing that there is only one            hydrophobic separation handle within the molecule or the            hydrophobicity of either of these groups is substantially            higher than the other;

-   2) The modified polymer of claim 1, wherein the functional group M    is selected from the group consisting of hydroxyl, amine, thiol,    carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl,    azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide,    dithiopyridine, iodoacetamide, biotin or a fluorophore.

-   3) The modified polymer of claim 1, wherein the group K is selected    from the group consisting of methyl, ethyl, propyl, isopropyl,    butyl, isobutyl, sec-butyl, tert-butyl, hexyl, or a residue from    diethylene glycol, triethylene glycol, tetraethylene glycol or    hexaethylene glycol.

-   4) The modified polymer of claim 1, wherein the group L is C₁-C₁₂    alkyl or substituted alkyl.

-   5) The modified polymer of claim 1, wherein the group R is selected    from trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,    FMOC, trifluoroacetyl, acetal, cyclic acetal or combinations thereof

-   6) The modified polymer of claim 1, wherein, for the functional    group M being carboxyl, the group R is selected from the group    consisting of chlorotrityl, trityl, N-hydroxysuccinimidyl,    p-nitrophenyl, pentachlorophenyl, simple non-activating alkyls    selected from the group of C₁-C₁₈ alkyls or none.

-   7) The modified polymer of claim 1, wherein the non peptidic and non    nucleotidic polymer backbone is selected from the group consisting    of poly(alkylene glycol), poly(oxyethylated polyol), poly(olefinic    alcohol), poly(α-hydroxy acid), poly(vinyl alcohol), polyoxazoline,    and copolymers and mixtures thereof

-   8) The modified polymer of claim 1, wherein the non peptidic and non    nucleotidic polymer backbone is poly(ethylene glycol).

-   9) The modified polymer of claim 8, wherein the poly(ethylene    glycol) has an average molecular weight from about 500 Da to about    100000 Da.

-   10) An essentially pure, linear form of modified polymer of claim 1,    wherein the two termini of the polymer are modified    non-symmetrically with two different functional groups and wherein    the two functional groups are linked to the polymer as in the    structure:

wherein:

-   -   M and M₁ are two different functional groups reactive with a        biologically active molecule or detectable functional groups;    -   E and E₁ are independently oxygen or sulfur;    -   The pair Z₁ and Z₂ are independent from the pair Z₃ and Z₄ and        both Z₁ and Z₂ may be O, but when Z₁ is NH then Z₂ is O, and        when Z₂ is NH then Z₁ is O, similarly for a pair of Z₃ and        Z₄—both Z₃ and Z₄ may be O, but when Z₃ is NH then Z₄ is O, and        when Z₄ is NH then Z₃ is O;    -   L and L₁ are independently selected from the group consisting of        linear alkyl, branched alkyl, alkyloxyalkyl, oligomeric form of        alkyloxyalkyl, aryl and substituted aryl;    -   R and R₁ are independently protecting groups, activating groups        or none;    -   K and K₁ are independently selected from the group consisting of        linear alkyl, branched alkyl, alkyloxyalkyl, or oligomeric form        of alkyloxyalkyl;    -   G and G₁ are independently selected from the group consisting of        none, an alkoxy, trityloxy or substituted trityloxy group;    -   L-M-R and L₁-M₁-R₁ fragments are linked to the respective        terminus of the said polymer via phosphotriester,        thiophosphotriester or amidophosphotriester bonds;

-   11) The modified polymer of claim 10, wherein the two different    functional groups M and M₁ are selected independently from the group    consisting of hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal,    dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl    sulfone, hydrazide, aminoxy, maleimide, dithiopyridine,    iodoacetamide, biotin or a fluorophore.

-   12) The modified polymer of claim 10, wherein the group K is    selected from the group consisting of methyl, ethyl, propyl,    isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, hexyl, or a    residue from diethylene glycol, triethylene glycol, tetraethylene    glycol or hexaethylene glycol.

-   13) The modified polymer of claim 10, wherein the group L is C₁-C₁₂    alkyl or substituted alkyl.

-   14) The modified polymer of claim 10, wherein the group R is    selected from trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,    alkoxypixyl, FMOC, trifluoroacetyl, acetal, cyclic acetal or    combinations of thereof.

-   15) The modified polymer of claim 10, wherein, for the functional    group M being carboxyl, the group R is selected from the group    consisting of chlorotrityl, trityl, N-hydroxysuccinimidyl,    p-nitrophenyl, pentachlorophenyl, simple non-activating alkyls    selected from the group of C₁-C₁₈ alkyls or none.

-   16) The modified polymer of claim 10, wherein the polymer is    poly(ethylene glycol).

-   17) The modified polymer of claim 16, wherein the poly(ethylene    glycol) has an average molecular weight from about 500 Da to about    100000 Da.

-   18) An essentially pure, linear form of modified polymer of claim 1,    wherein the two termini of the polymer are modified    non-symmetrically with two different functional groups and wherein    the two functional groups are linked to the polymer as in the    structure:

wherein:

-   -   M and M₂ are two different functional groups reactive with a        biologically active molecules or M is a detectable functional        group;    -   E is O or S;    -   K is selected from the group consisting of linear alkyl,        branched alkyl, alkyloxyalkyl, or oligomeric form of        alkyloxyalkyl;    -   G is none or is selected from the group consisting of an alkoxy,        trityloxy, monoalkoxy substituted trityloxy group or dialkoxy        substituted trityloxy group;    -   Z₁ and Z₂ are O or NH in such way that both Z₁ and Z₂ may be O,        but when Z₁ is NH then Z₂ is O, and when Z₂ is NH then Z₁ is O;    -   L is selected from the group consisting of linear alkyl,        branched alkyl, alkyloxyalkyl, oligomeric form of alkyloxyalkyl,        aryl and substituted aryl;    -   R is a protecting group, activating group, hydrogen or none;    -   L-M-R fragment is linked to the first terminus of the said        polymer via phosphotriester, thiophosphtriester or        amidophosphotriester;    -   M₂ is O, S or NH;    -   M₂ is linked directly to the second terminus of the polymer, and        not via phosphotriester, thiophosphtriester or        amidophosphotriester;    -   R₂ is a protecting group or none.

-   19) The modified polymer of claim 18, wherein the functional group M    is selected from the group consisting of hydroxyl, amine, thiol,    carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl,    azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide,    dithiopyridine, iodoacetamide or biotin.

-   20) The modified polymer of claim 18, wherein the group R₂ is    selected from the group consisting of trityl, monoalkoxytrityl,    dialkoxytrityl, pixyl, alkoxypixyl, FMOC, alkylcarboxyl, benzoyl,    tetrahydropyranyl, methyl or none.

-   21) The modified polymer of claim 18, wherein the polymer is    poly(ethylene glycol).

-   22) The conjugate of any polymer of claims 1 to 18 with a    biologically active molecule wherein said biologically active    molecule is selected from the group consisting of enzymes, peptides,    polypeptides, nucleotides, oligonucleotides, polynucleotides and low    molecular weight drugs.

-   23) A method of synthesizing a substantially pure, water soluble    polymer of claim 1, said method comprising the steps of:    -   a) contacting the water soluble polymer, of non-peptidic and        non-nucleotidic type, and having linear or branched polymer        backbone, and containing from 2 and up to 100 termini, in a        water free solvent, with a selected modifying reagent in form of        the phosphoramidite derivative as in the structure:

-   -   -   wherein:        -   R₃ and R₄ are isopropyls or are a part of of morpholine            ring.

    -   b) starting reaction by addition of an activating reagent.

    -   c) oxidation of P⁺³ to P⁺⁵ by addition of an oxidizing reagent.

    -   d) optional chromatographic purification of the protected        polymer.

    -   e) removal of the protecting groups.

-   24) Method of claim 23 wherein the activating reagent is selected    from tetrazole, 2-ethylthiotetrazole, 2-bezylthiotetrazole,    4,5-dicyanoimidazole, “Activator 42”, pyridinium hydrochloride or    pyridinium trifluoroacetate

-   25) Method of claim 23 wherein the oxidizing reagent is selected    from a group consisting of iodine, water peroxide, t-butyl hydrogen    peroxide, acetone peroxide, sulfur and thiuram disulfide.

-   26) Method of claim 23, wherein the ratio between the polymer and    the phosphoramidite is from 1:1 to 10:1 in order to facilitate    formation of monosubstituted product.

-   27) A method of synthesizing a substantially pure, water soluble    polymer of claim 10, said method comprising the steps of:    -   a) reacting the water soluble polymer, of non-peptidic and        non-nucleotidic type, having linear polymer backbone, and        containing two reactive termini, in a water free solvent, with a        selected first modifying reagent in form of the phosphoramidite        derivative, under conditions that facilitate formation of        monoderivatized product.    -   b) chromatographic isolation of the monoderivatized polymer.    -   c) reacting the monoderivatized product with a second modifying        reagent in the form of phosphoramidite derivative, under        conditions that facilitate the quantitative conversion to the        double modified polymer.    -   d) isolation of the double modified polymer by precipitation or        crystallization.

-   28) A method of synthesizing a substantially pure, water soluble    polymer of claim 18, said method comprising the steps of:    -   a) reacting the substantially pure, linear polymer, substituted        at the first terminus with a function R₂-M₂, with a selected        modifying reagent in form of the phosphoramidite derivative        under conditions facilitating the quantitative conversion of the        mono substituted polymer to the double modified polymer.    -   b) isolation of the double modified polymer by precipitation or        crystallization.

-   29) The use of any material of claim 1 to claim 28 for formation of    a conjugate between this material and a biologically active    molecule, wherein said biologically active molecule is selected from    the group consisting of enzymes, peptides, polypeptides,    nucleotides, oligonucleotides, polynucleotides and low molecular    weight drugs.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A conjugate, or a pharmaceutically acceptablesalt thereof, comprising a water-soluble, non-peptidic, andnon-nucleotidic polymer backbone having at least one terminus covalentlybonded to a structure of formula (9):

or a salt thereof, wherein: A is the point of covalent bonding to theterminus of the polymer backbone, wherein the polymer backbone ispoly(ethylene glycol), wherein said poly(ethylene glycol) has an averagemolecular weight from about 500 Da to about 100,000 Da; E is O; K isselected from the group consisting of: alkylene, alkyleneoxyalkylene,and oligomeric alkyleneoxyalkylene; G is selected from the groupconsisting of: hydrogen, alkoxy, and a hydrophobic separation handle; Z¹and Z² are O; L is selected from the group consisting of: a divalentradical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomericalkyleneoxyalkylene, and unsubstituted and substituted arylene; R¹ isabsent or a hydrophobic separation handle, wherein only one of R¹ and Gcan be a hydrophobic separation handle; L² is a covalent linking moietybetween L on the polymer backbone and B; and B is insulin or aderivative thereof.
 2. The conjugate of claim 1, wherein only onetermini of the polymer backbone is covalently bonded to the structure offormula (9).
 3. The conjugate of claim 1, wherein the polymer backbonehas two termini.
 4. The conjugate of claim 3, wherein only one terminiof the polymer backbone is covalently bonded to the structure of formula(9).
 5. The conjugate of claim 3, wherein both termini of the polymerbackbone are covalently bonded to a structure of formula (9).
 6. Theconjugate of claim 1, wherein K is selected from the group consistingof: methylene, ethylene, propylene, isopropylene, butylene, isobutylene,sec-butylene, tert-butylene, and hexylene, or a residue from diethyleneglycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol.7. The conjugate of claim 1, wherein G is a substituted or unsubstitutedtrityloxy.
 8. The conjugate of claim 1, wherein L is a substituted orunsubstituted C₁-C₁₂ alkylene.
 9. A conjugate, or a pharmaceuticallyacceptable salt thereof, comprising a structure of formula (10):

or a salt form thereof, wherein: polymer is poly(ethylene glycol),wherein said poly(ethylene glycol) has an average molecular weight fromabout 500 Da to about 100,000 Da, wherein each linking group is bondedat a different terminus of said polymer; E and E¹ are independently O; Kand K₁ are independently selected from the group consisting of:alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G andG₁ are independently absent or are selected from the group consistingof: alkoxy and a hydrophobic separation handle; each pair of Z¹ and Z²and Z³ and Z⁴ are O, L and L¹ are independently selected from the groupconsisting of: a divalent radical of a nucleoside, alkylene,alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstitutedand substituted arylene; L² is a covalent linking moiety between L onthe polymer backbone and B; L³ is a covalent linking moiety between L onthe polymer backbone and B¹; and B and B¹ are independently insulin, aderivative of insulin, a biologic other than insulin, a drug, adetectable group, a separation moiety, wherein at least one of B and B¹is insulin or a derivative of insulin.
 10. A conjugate, or apharmaceutically acceptable salt thereof, comprising a compound offormula (11):

or a salt form thereof, wherein: polymer is poly(ethylene glycol),wherein said poly(ethylene glycol) has an average molecular weight fromabout 500 Da to about 100,000 Da, wherein L⁴ and the phosphonate-derivedfunctional group are bonded at a different terminus of said polymer; Eand E¹ are O; K is selected from the group consisting of: alkylene,alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G is selectedfrom the group consisting of: hydrogen, alkoxy, and a hydrophobicseparation handle; Z¹ and Z² are independently O; L is selected from thegroup consisting of: a divalent radical of nucleoside, alkylene,alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstitutedand substituted arylene; L² is a covalent linking moiety between L onthe polymer backbone and B; L⁴ is a covalent linking moiety between L onthe polymer backbone and B¹; and B and B¹ are independently insulin, aderivative of insulin, a biologic other than insulin, a drug, adetectable group, a separation moiety, wherein at least one of B and B¹is insulin or a derivative of insulin.
 11. A composition comprising theconjugate of any one of claims 1, 9, and 10, and a pharmaceuticallyacceptable excipient.
 12. The composition of claim 11, wherein B isinsulin.
 13. The composition of claim 11, wherein B¹ is insulin.
 14. Amethod of treating a patient diagnosed with diabetes, said methodcomprising administering to said patient an effective amount of theconjugate of claim
 1. 15. The method of claim 14, wherein B is insulin.16. The method of claim 6, wherein G is hydrogen.